Formulations of guanylate cyclase c agonists and methods of use

ABSTRACT

The invention provides novel formulations of guanylate cyclase-C (“GCC”) agonist peptides and methods for their use in the treatment of gastrointestinal diseases and disorders, including gastrointestinal cancer. The GCC agonist formulations of the invention can be administered either alone or in combination with one or more additional therapeutic agents, preferably an inhibitor of cGMP-dependent phosphodiesterase or a laxative.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/301,812, filed Jun. 11, 2014 which is a continuation of U.S. Utilityapplication Ser. No. 12/630,565, filed on Dec. 3, 2009, now abandonedand claims the benefit of priority to U.S. Provisional Application No.61/119,521 filed on Dec. 3, 2008, the contents of which are incorporatedby reference in their entireties.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is SYPA_007_C02US_ST25.txt. The text file is about116 KB, was created on Jun. 26, 2018, and is being submittedelectronically via EFS-Web.

FIELD OF THE INVENTION

The present invention relates to novel formulations of guanylate cyclaseC agonists which are optimized for delivery to specific regions of thegastrointestinal tract and are useful for the treatment and preventionof gastrointestinal diseases and disorders.

BACKGROUND OF THE INVENTION

Guanylate cyclase C is a transmembrane form of guanylate cyclase that isexpressed on various cells, including gastrointestinal epithelial cells(reviewed in Vaandrager 2002 Mol. Cell. Biochem. 230:73-83). It wasoriginally discovered as the intestinal receptor for the heat-stabletoxin (ST) peptides secreted by enteric bacteria and which causediarrhea. The ST peptides share a similar primary amino acid structurewith two peptides isolated from intestinal mucosa and urine, guanylinand uroguanylin (Currie, et al., Proc. Nat'l Acad. Sci. USA 89:947-951(1992); Hamra, et al., Proc. Nat'l Acad. Sci. USA 90:10464-10468 (1993);Forte, L., Reg. Pept. 81:25-39 (1999); Schulz, et al., Cell 63:941-948(1990); Guba, et al., Gastroenterology 111:1558-1568 (1996); Joo, etal., Am. J. Physiol. 274:G633-G644 (1998)).

In the intestines, guanylin and uroguanylin act as regulators of fluidand electrolyte balance. In response to high oral salt intake, thesepeptides are released into the intestinal lumen where they bind toguanylate cyclase C localized on the luminal membrane of enterocytes(simple columnar epithelial cells of the small intestines and colon).The binding of the guanylin peptides to guanylate cyclase C induceselectrolyte and water excretion into the intestinal lumen via a complexintracellular signaling cascade that is initiated by an increase incyclic guanosine monophosphate (cGMP).

The cGMP-mediated signaling that is initiated by the guanylin peptidesis critical for the normal functioning of the gut. Any abnormality inthis process could lead to gastrointestinal disorders such as irritablebowel syndrome (IBS) and inflammatory bowel diseases. Inflammatory boweldisease is a general name given to a group of disorders that cause theintestines to become inflamed, characterized by red and swollen tissue.Examples include ulcerative colitis and Crohn's disease. Crohn's diseaseis a serious inflammatory disease that predominantly affects the ileumand colon, but can also occur in other sections of the gastrointestinaltract. Ulcerative colitis is exclusively an inflammatory disease of thecolon, the large intestine. Unlike Crohn's disease, in which all layersof the intestine are involved, and in which there can be normal healthybowel in between patches of diseased bowel, ulcerative colitis affectsonly the innermost lining (mucosa) of the colon in a continuous manner.Depending on which portion of the gastrointestinal tract is involved,Crohn's disease may be referred to as ileitis, regional enteritis,colitis, etc. Crohn's disease and ulcerative colitis differ from spasticcolon or irritable bowel syndrome, which are motility disorders of thegastrointestinal tract. Gastrointestinal inflammation can be a chroniccondition. It is estimated that as many as 1,000,000 Americans areafflicted with inflammatory bowel disease, with male and female patientsappearing to be equally affected. Most cases are diagnosed before age30, but the disease can occur in the sixth, seventh, and later decadesof life.

IBS and chronic idiopathic constipation are pathological conditions thatcan cause a great deal of intestinal discomfort and distress but unlikethe inflammatory bowel diseases, IBS does not cause the seriousinflammation or changes in bowel tissue and it is not thought toincrease the risk of colorectal cancer. In the past, inflammatory boweldisease, celiac disease and IBS were regarded as completely separatedisorders. Now, with the description of inflammation, albeit low-grade,in IBS, and of symptom overlap between IBS and celiac disease, thiscontention has come under question. Acute bacterial gastroenteritis isthe strongest risk factor identified to date for the subsequentdevelopment of postinfective irritable bowel syndrome. Clinical riskfactors include prolonged acute illness and the absence of vomiting. Agenetically determined susceptibility to inflammatory stimuli may alsobe a risk factor for irritable bowel syndrome. The underlyingpathophysiology indicates increased intestinal permeability andlow-grade inflammation, as well as altered motility and visceralsensitivity. Serotonin (5-hydroxytryptamine [5-HT]) is a key modulatorof gut function and is known to play a major role in pathophysiology ofIBS. The activity of 5-HT is regulated by cGMP.

While the precise causes of IBS and inflammatory bowel diseases (IBD)are not known, a disruption in the process of continual renewal of thegastrointestinal mucosa may contribute to disease pathology in IBD andaggravate IBS. The renewal process of the gastrointestinal lining is anefficient and dynamic process involving the continual proliferation andreplenishment of unwanted damaged cells. Proliferation rates of cellslining the gastrointestinal mucosa are very high, second only to thehematopoietic system. Gastrointestinal homeostasis depends on both theproliferation and programmed cellular death (apoptosis) of epithelialcells lining the gut mucosa. Cells are continually lost from the villusinto the lumen of the gut and are replenished at a substantially equalrate by the proliferation of cells in the crypts, followed by theirupward movement to the villus. The rates of cell proliferation andapoptosis in the gut epithelium can be increased or decreased in avariety of circumstances, e.g., in response to physiological stimulisuch as aging, inflammatory signals, hormones, peptides, growth factors,chemicals and dietary habits. In addition, an enhanced proliferationrate is frequently associated with a reduction in turnover time and anexpansion of the proliferative zone. The proliferation index is muchhigher in pathological states such as ulcerative colitis and othergastrointestinal disorders. Intestinal hyperplasia is a major promoterof gastrointestinal inflammation. Apoptosis and cell proliferationtogether regulate cell number and determine the proliferation index.Reduced rates of apoptosis are often associated with abnormal growth,inflammation, and neoplastic transformation. Thus, both increasedproliferation and/or reduced cell death may increase the proliferationindex of intestinal tissue, which may in turn lead to gastrointestinalinflammatory diseases.

In addition to a role for uroguanylin and guanylin as modulators ofintestinal fluid and ion secretion, these peptides may also be involvedin the continual renewal of gastrointestinal mucosa by maintaining thebalance between proliferation and apoptosis. For example, uroguanylinand guanylin peptides appear to promote apoptosis by controllingcellular ion flux. Given the prevalence of inflammatory conditions inWestern societies a need exists to improve the treatment options forinflammatory conditions, particularly of the gastrointestinal tract.

SUMMARY OF THE INVENTION

The present invention provides novel formulations of guanylate cyclase Cagonists (“GCC agonists”) which are optimized for the targeted deliveryof the agonist to a specific portion of the gastrointestinal tract, forexample, to the small intestines, preferably to the duodenum or jejunum,or to the distal small intestines or the large intestines, preferablythe ileum, terminal ileum, or ascending colon. The formulationsoptimized for delivery of a GCC agonist to the duodenum or jejunum areparticularly useful for the treatment or prevention of a disease ordisorder selected from the group consisting of irritable bowel syndrome(preferably constipation predominant) non-ulcer dyspepsia, chronicintestinal pseudo-obstruction, functional dyspepsia, colonicpseudo-obstruction, duodenogastric reflux, gastro esophageal refluxdisease, chronic idiopathic constipation, gastroparesis, heartburn,gastric cancer, and H. pylori infection. The formulations optimized fordelivery of a GCC agonist to the ileum, terminal ileum, or ascendingcolon are particularly useful for the treatment or prevention of adisease or disorder selected from the group consisting of ileitis (e.g.,post-operative ileitis), Crohn's disease, ulcerative colitis, terminalileitis, and colon cancer.

The targeted GCC agonist formulations of the invention offer severaladvantages over other formulations, especially conventional oralformulations. Because GCC agonists can potentially act throughout thegastrointestinal tract, conventional oral formulations intended to treatIBD, for instance, may exhibit side effects due to the activity of theGCC agonist in non-target tissues. One such side effect is diarrhea,which could interfere with treatment by a GCC agonist of GI diseasessuch as ulcerative colitis and Crohn's disease. Conventional oralformulations also suffer from degradation or aggregation of the GCCagonist in the stomach due to the low pH environment (Marx et al., 1998Peptide Res. 52:229-240; Chino et al., 1998 FEBS Let. 421:27-31). Incontrast, the GCC agonist formulations of the invention are optimizedfor the release of the GCC agonist to the target tissue, either thesmall intestines or the large intestines, depending on the disease ordisorder to be treated. Such formulations minimize exposure of the GCCagonist peptide to stomach acidity, thereby reducing or eliminating thedegradation and aggregation that occur under low pH conditions. Otheradvantages of the GCC agonist formulations of the invention includefewer side effects caused by unwanted GCC activity in non-targettissues. In addition, the GCC agonists formulated according to theinvention may be given at a lower effective dose than a convential oraldosage form. In other embodiments, the GCC agonist formulation of theinvention delivers a higher effective dose to the target tissue than aconvential oral dosage form with reduced side effects compared to aconvential oral dosage form.

In certain embodiments, the GCC agonists are analogs of uroguanylin andbacterial ST peptides. In preferred embodiments, the analogs havesuperior properties compared to the naturally occurring or “wild-type”peptides. Examples of such superior properties include a high resistanceto degradation at the N-terminus and C-terminus from carboxypeptidases,aminopeptidases, and/or by other proteolytic enzymes present in thestimulated human intestinal juices and human gastric juices. Examples ofGCC agonists that can be used in the formulations and methods of theinvention are described in more detail below.

In one embodiment, the GCC agonist formulation comprises (1) a core,which contains at least one GCC agonist peptide, and (2) one or moretargeting materials selected from the group consisting of a pH-dependentpolymer, a swellable polymer, and a degradable composition, wherein theGCC agonist peptide is selected from the group consisting of SEQ ID NOs:1-249. In a preferred embodiment, the GCC agonist peptide is selectedfrom the group consisting of SEQ ID NOs: 1, 8, 9, 55 or 56. In oneembodiment, the GCC agonist peptide is selected from the groupconsisting of SEQ ID NOs: 1 and 9. In certain embodiments, the one ormore targeting materials form one or more layers around the core. Incertain embodiments, at least one targeting material forms a matrix withthe GCC agonist peptide of the core. Preferably, the formulation is fororal administration.

In one embodiment, the GCC agonist formulation is optimized for deliveryof a GCC agonist to the duodenum or jejunum and comprises one or more pHdependent polymers which degrade in a pH range of 4.5 to 5.5, whereinthe pH dependent polymers form one or more layers around the core.

In one embodiment, the GCC agonist formulation is optimized for deliveryof a GCC agonist to the ileum, terminal ileum, or ascending colon andcomprises one or more pH dependent polymers which degrade in a pH rangeof 5.5 to 6.5 or in a pH range of 6.5 to 7.5, wherein the pH dependentpolymers form one or more layers around the core. In one embodiment, theone or more pH dependent polymers degrade at pH above 5.5. In anotherembodiment, the one or more pH dependent polymers degrade at pH above 7.In one embodiment, the formulation further comprises a swellable polymerinterposed between two layers of pH dependent polymers. Preferably, theswellable polymer is selected from the group consisting of an acryliccopolymer, polyvinylacetate, and cellulose derivatives. In oneembodiment, the swellable polymer is an acrylic copolymer selected fromthe group consisting of EUDRAGIT® RL, EUDRAGIT® RS, and EUDRAGIT® NE. Inone embodiment, the formulation further comprises a pore forming agent.In specific embodiments, the pore forming agent is selected from thegroup consisting of saccharose, sodium chloride, potassium chloride,polyvinylpyrrolidone, polyethyleneglycol, water soluble organic acids,sugars and sugar alcohol.

The pH dependent polymers for use in the formulations of the inventonare preferably selected from the group consisting of a methacrylic acidcopolymer, a polyvinyl acetate phthalate, a hydroxypropylmethylcellulosephthalate, a cellulose acetate trimelliate, a cellulose acetatephthalate, or a hydroxypropyl methyl cellulose acetate succinate. In oneembodiment, at least one of the pH dependent polymers is a methacrylicacid copolymer. In a preferred embodiment, the methacrylic acidcopolymer is selected from among the EUDRAGIT® polymers. In a particularembodiment, the EUDRAGIT® polymer is selected from among the groupconsisting of EUDRAGIT® L100, EUDRAGIT® L-30D, EUDRAGIT® S100, EUDRAGIT®FS 30D, and EUDRAGIT® L100-55, and combinations thereof.

In one embodiment, the GCC agonist formulation comprises a degradablecomposition. In certain embodiments, the degradable composition isselected from the group consisting of amylase, chitosan, chondroitinsulfate, cyclodextrin, dextran, guar gum, pectin, and xylan. Preferably,the degradable composition is coated with a material selected from thegroup consisting of cellulose acetate phthalate, hydroxy propyl methylcellulose acetate succinate, EUDRAGIT® L100 and EUDRAGIT® L30D-55.

In another embodiment, the degradable composition is a carrier moleculelinked to the GCC agonist by a covalent bond, wherein the covalent bondis stable in the stomach and small intestines but labile in the lowergastrointestinal tract, especially the colon. Preferably, the covalentbond is an azo bond or a glycosidic bond. In a specific embodiment, thecarrier molecule is selected from the group consisting of a glucuronide,a cyclodextrin, a dextran ester, or a polar amino acid.

The invention also provides methods for treating or preventing agastrointestinal disease or disorder in a subject in need thereof,comprising administering to the subject a GCC agonist formulationcomprising (1) a core, which contains at least one GCC agonist peptide,and (2) one or more targeting materials selected from the groupconsisting of a pH-dependent polymer, a swellable polymer, and adegradable composition, wherein the GCC agonist peptide is selected fromthe group consisting of SEQ ID NOs: 1-249. In certain embodiments, theone or more targeting materials form one or more layers around the core.In certain embodiments, at least one targeting material forms a matrixwith the GCC agonist peptide of the core. Preferably, the formulation isfor oral administration.

In one embodiment, the formulation comprises one or more pH dependentpolymers which degrade in a pH range of 4.5 to 5.5 and thegastrointestinal disease or disorder is selected from the groupconsisting of irritable bowel syndrome (preferably constipationpredominant), non-ulcer dyspepsia, chronic intestinalpseudo-obstruction, functional dyspepsia, colonic pseudo-obstruction,duodenogastric reflux, gastro esophageal reflux disease, chronicidiopathic constipation, gastroparesis, heartburn, gastric cancer, andH. pylori infection. In one embodiment, the gastrointestinal disease ordisorder is selected from the group consisting of chronic idiopathicconstipation and irritable bowel syndrome.

In one embodiment, the formulation comprises one or more pH dependentpolymers which degrade in a pH range of 6.5 to 7.5 and thegastrointestinal disease or disorder is selected from the groupconsisting of ileitis (post-operative ileitis), Crohn's disease,ulcerative colitis, terminal ileitis, and colon cancer. In oneembodiment, the gastrointestinal disease or disorder is selected fromthe group consisting of ulcerative colitis and Crohn's disease.

In a preferred embodiment, the method for treating or preventing agastrointestinal disease or disorder in a subject in need thereofcomprises administering to the subject a GCC agonist formulationcomprising a GCC agonist peptide selected from the group consisting ofSEQ ID NOs: 1, 8, 9, 55 or 56. In one embodiment, the GCC agonistpeptide is selected from the group consisting of SEQ ID NOs: 1 and 9.

In one embodiment, the method further comprises administering to thesubject an effective amount of an inhibitor of a cGMP-specificphosphodiesterase. In a specific embodiment, the cGMP-dependentphosphodiesterase inhibitor is selected from the group consisting ofsuldinac sulfone, zaprinast, and motapizone, vardenifil, and suldenifil.

In one embodiment, the method further comprises administering to thesubject an effective amount of at least one laxative. In one embodiment,the at least one laxative is selected from the group consisting ofSENNA, MIRALAX, PEG, or calcium polycarbophil.

In one embodiment, the method further comprises administering to thesubject an effective amount of at least one anti-inflammatory agent.

In a preferred embodiment, the subject is a human.

Other features and advantages of the invention will be apparent from andare encompassed by the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A: Biological activity of SP-304 incubated for varying lengths oftime in simulated gastric fluid (SGF). The biological activity of SP-304was determined by measuring its ability to stimulate cGMP synthesis inT84 cells. Samples incubated for 0, 15, 30, 60, 90 and 120 minutes,respectively, were collected and analyzed for biological activity—withactivity of the 0 min sample defined as 100% biological activity. Theactivities of the other samples are shown as percentage activityrelative to the 0 min activity sample. Data points shown are the averageof triplicate measurements ±SD.

FIG. 1B: Schematic representation of HPLC chromatographic analyses ofSP-304 samples after incubation with SGF for 0 and 120 min,respectively. The arrows show the elution position of SP-304.

FIG. 2A: Biological activity of SP-304 incubated for varying lengths oftime in simulated intestinal fluid (SIF). SP-304 samples were incubatedfor 0, 30, 60, 90, 150, and 300 min, respectively, and then tested fortheir ability to stimulate cGMP synthesis in T84 cells. The cGMPstimulation activity in the sample at 0 min of incubation time in SIFwas taken as 100% biological activity. The activities of the otherSP-304 incubation samples were calculated as the percentage of activityrelative to that of the 0 min sample. Data points shown are the averageof triplicate measurements ±SD.

FIG. 2B: HPLC chromatographic spectra of SP-304 samples after incubationwith: (A) heat-inactivated SIF for 300 minutes, or (B) SIF for 120minutes. The arrows show the elution position of SP-304. The treatmentwith SIF completely eliminated SP-304 after a 2-hr incubation and apeptide signal appeared, eluting at 9.4 min, as indicated by *.

FIG. 3: Schematic representation of possible degradation products ofSP-304.

FIG. 4: Biological activity of truncated peptides of 16-mer SP-304, asmeasured by stimulation of cGMP synthesis in T84 cells. SP-338 15-merpeptide is indentical to SP-304 except that it lacks Leu at theC-terminus. SP-327, SP-329 and SP-331 lack Leu at their C-terminirelative to their corresponding parents, SP-326, SP-328 and SP-330,respectively. Data points shown are the average of duplicatemeasurements.

FIG. 5: Stimulation of cGMP synthesis in T84 cells by SP-304 and similaranalogs. T84 cells were exposed to test peptides for 30 min and celllysates were then used to determine intracellular cGMP levels. Datapoints shown are the average of triplicate measurements ±SD.

FIG. 6: Stimulation of cGMP synthesis in T84 cells by SP-339(linaclotide) and other linaclotide analogs. T84 cells were exposed totest peptides for 30 min and cell lysates were then used to determineintracellular cGMP levels. Data points shown are the average oftriplicate measurements ±SD.

FIG. 7A: Biological activity of SP-333 incubated for varying lengths oftime in simulated intestinal fluid (SIF). SP-333 samples incubated for0, 5, 10, 30, 60, and 120 min, respectively, were tested for theirability to stimulate cGMP synthesis in T84 cells. The control samplemarked as C120 was produced by incubating SP-333 with heat inactivatedSIF for 120 min. Samples from the incubations were removed and heated at95° C. for 5 min to inactivate digestive enzymes and then used tostimulate cyclic GMP synthesis in T84 cells. The cGMP stimulationactivity in the sample at 0 min of incubation time in SIF was taken as100% biological activity. The activities of the other SP-304 incubationsamples were calculated as the percentage of activity relative to thatof the 0 min sample. Data points shown are the average of triplicatemeasurements ±SD.

FIG. 7B: Biological activity of SP-332 incubated for varying lengths oftime in simulated intestinal fluid (SIF). SP-333 samples incubated for0, 5, 10, 30, 60, and 120 min, respectively, were tested for theirability to stimulate cGMP synthesis in T84 cells. The control samplemarked as C120 was produced by incubating SP-332 with heat inactivatedSIF for 120 min. Samples from the incubations were removed and heated at95° C. for 5 min to inactivate digestive enzymes and then used tostimulate cyclic GMP synthesis in T84 cells. The cGMP stimulationactivity in the sample at 0 min of incubation time in SIF was taken as100% biological activity. The activities of the other SP-304 incubationsamples were calculated as the percentage of activity relative to thatof the 0 min sample. Data points shown are the average of triplicatemeasurements ±SD.

FIG. 7C: Biological activity of SP-304 incubated for varying lengths oftime in simulated intestinal fluid (SIF). SP-304 samples incubated for0, 10, and 60 min, respectively, were tested for their ability tostimulate cGMP synthesis in T84 cells. The control samples marked as C0and C60 were produced by incubating SP-304 with heat inactivated SIF for0 and 60 min, respectively. Samples from the incubations were removedand heated at 95° C. for 5 min to inactivate digestive enzymes and thenused to stimulate cyclic GMP synthesis in T84 cells. The cGMPstimulation activity in the sample at 0 min of incubation time in SIFwas taken as 100% biological activity. The activities of the otherSP-304 incubation samples were calculated as the percentage of activityrelative to that of the 0 min sample. Data points shown are the averageof triplicate measurements ±SD.

FIGS. 7D-1-7D-2: HPLC chromatograms of SP-304 incubated for 0 min in SIF(FIG. 7D-1) and 60 min in SIF (FIG. 7D-2), respectively. The arrowsindicate the elution positions of the parent SP-304 peptides. The dataclearly show that the SP-304 peak eluting at 14.3 min completelyvanished and two new peaks emerged at 7.4 and 10.3 minutes. These newpeptide peaks represent degradation products of SP-304.

FIGS. 7E-1-7-E2: HPLC chromatograms of SP-332 incubated for 0 min in SIF(FIG. 7E-1) and 120 min in SIF (FIG. 7E-2), respectively. The arrowsindicate the elution positions of the parent SP-332 peptides. The datashow that the peptide SP-332 eluting at 14.8 minutes remained intactfollowing incubation with SIF for 120 min, suggesting that SP-332 isresistant to proteolysis by proteases present in SIF.

FIGS. 7F-1-7F-2: HPLC chromatograms of SP-333 incubated for 0 min in SIF(FIG. 7F-1) and 120 min in SIF (FIG. 7F-2), respectively. The arrowsindicate the elution positions of the parent SP-333 peptides. The datashow that peptide SP-333, eluting at 14.8 minutes remained intactfollowing incubation with SIF, suggesting that SP-333 is resistant toproteolysis by proteases present in SIF during the 120 minute incubationperiod.

FIGS. 7G-1-7G-5: Incubation of SP-333 with SIF for 2, 6, 12 and 24 hrs.Effect of SIF digestion on the activity of SP-333 in T84 stimualationassay was determined (FIG. 7G-1) and samples were also analyzed by HPLC(FIGS. 7G2-5). The elution positions of the parent peptide and itsmetabolites are indicated by arrows.

FIG. 8: Stimulation of cGMP synthesis in T84 cells by the peggylatedanalogs of SP-333. T84 cells were exposed to the indicated peptides for30 min and cell lysates were used to determine intracellular cGMPlevels. Data points shown are the average of triplicate measurements±SD.

FIG. 9: Stimulation of cGMP synthesis in T84 cells by SP-304 (0.1 μM)either alone or in combination with the phosphodiesterase (PDE)inhibitors Sulindac Sulfone (100 μM) or Zaprinast (100 μM). T84 cellswere exposed to various treatments, as indicated, for 30 min and thecell lysates were used to determine the intracellular cGMP levels. Datapoints shown are the average of duplicate measurements.

FIG. 10: Stimulation of cGMP synthesis in T84 cells by SP-304 (0.1 or1.0 μM) either alone or in combination with incremental concentrationsof phosphodiesterase (PDE) inhibitors, as indicated. T84 cells wereexposed to various treatments, as indicated, for 30 min and the celllysates were used to determine the intracellular cGMP levels. Datapoints shown are the average of duplicate measurements.

FIG. 11: Stimulation of cGMP synthesis in T84 by SP-333 (0.1 or 1.0 μM)either alone or in combination with incremental concentrations ofZaprinast, as indicated. T84 cells were exposed to various treatments,as indicated, for 30 min and the cell lysates were used to determine theintracellular cGMP levels. Data points shown are the average ofduplicate measurements.

FIG. 12: Stimulation of cGMP synthesis in T84 by SP-333 (0.1 μM) eitheralone or in combination with incremental concentrations of SulindacSulfone, as indicated. T84 cells were exposed to various treatments, asindicated, for 30 min and the cell lysates were used to determine theintracellular cGMP levels. Data points shown are the average ofduplicate measurements.

FIG. 13: SP-304 treatment improved stool consistency and clearsTNBS-induced intestinal blockage in a TNBS-induced murine model ofcolitis.

FIG. 14: SP-304 treatment stimulated increased water flow in the lumenof the GI tract of cynomolgus monkeys.

FIG. 15A-B: The effect of SP-304 administration on stool consistency inhuman volunteers as judged by the Bristol Score of the first bowelmovement. Results from a phase 1, single-site, randomized, double-blind,placebo-controlled, single-, ascending-, oral-dose, sequential doseescalation study of SP-304 in fasted, healthy male and female subjects.A total of 9 cohorts utilizing 8 subjects per cohort (6 SP-304; 2placebos) were utilized, totaling 71 volunteers administered drug. Eachcohort was administered a single, oral dose or matching placeboadministered in 10-fold diluted phosphate buffered saline (PBS) (240mL). The nine cohort doses included 0.1, 0.3, 0.9, 2.7, 5.4, 8.1, 16.2,24.3 and 48.6 mg SP-304.

FIG. 16: The effect of SP-304 adminstration on average time to firststool through 24 hours post-dose in human volunteers. Results from aphase 1, single-site, randomized, double-blind, placebo-controlled,single-, ascending-, oral-dose, sequential dose escalation study ofSP-304 in fasted, healthy male and female subjects. A total of 9 cohortsutilizing 8 subjects per cohort (6 SP-304; 2 placebos) were utilized,totaling 71 volunteers administered drug. Each cohort was administered asingle, oral dose or matching placebo administered in 10-fold dilutedphosphate buffered saline (PBS) (240 mL). The nine cohort doses included0.1, 0.3, 0.9, 2.7, 5.4, 8.1, 16.2, 24.3 and 48.6 mg SP-304.

FIGS. 17A-B: SP-304 exhibited superior activity compared toSulfasalazine to ameliorate inflammation in DSS-induced colitis in BDF-1mice.

FIG. 18: SP-304 showed superior activity compared to Sulfasalazine toameliorate inflammation in TNBS-induced colitis in BDF-1 mice.

FIG. 19: Determination of SP-304 dose that produced diarrhea in monkeys.Two groups of monkeys, male and female, were treated with a single doseof SP-304 per day continuously for 28 days (for each group, 0 and 75mg/kg, n=5; 1, 10 mg/kg, n=4). Stool consistency and bowel frequencywere noted every day. Results are presented as cumulative score for 28days. The scoring used for stool consistency was as follows: 0: Nostool, 1: Normal stool, 2: Loose/mushy stool and 3: Diarrhea/waterystool.

FIG. 20: SP-304 formulated for pH dependent release. Gelatin capsuleswere filled with calculated quantity of SP-304 (10 mg/kg body weight).Capsules were coated with either EUDRAGIT® polymer L 30 D-55 (forrelease at pH greater than 5.5; Lot No. B081214690) or EUDRAGIT® polymerFSD (for release at pH greater than 7; Lot No. G090365030). One capsulesfrom each formulation were placed in a plastic tube containing 50 ml ofbuffer saline solution adjusted to either pH 5.7 or pH 7.2. Uncoatedgelatin capsules containing the same quantity of SP-304 were used ascontrols. All controls were incubated in a buffered saline solutionadjusted to pH 1.0. The plastic tubes were incubated on a rotatoryshaker in a 37° C. incubator. Samples (0.5 ml) were withdrawn from thetube at the indicated time intervals and immediately subjected to HPLCanalysis to determine the release of SP-304 in the solution. The capsulecoated with EUDRAGIT® polymer L 30 D-55 (for release at pH greater than5.5) was incubated at pH 2.5 for 60 min to mimic exposure to stomachacidity. The same capsule was removed and placed in buffer salinesolution (pH 5.7) and samples were withdrawn at different times for HPLCanalysis. Similarly, the capsule coated with EUDRAGIT® polymer FSD (forrelease at pH greater than 7) was sequentially incubated at pH 2.5 (60min), pH 5.5 (60 min) and then at pH 7.0 for the indicated time intervalfor sampling.

FIGS. 21A-C: Bioactivity of SP-304 formulated for pH dependent release.Gelatin capsules were filled with calculated quantity of SP-304 (10mg/kg body weight). Capsules were coated with EUDRAGIT® polymerfollowing the standard procedure for release at either pH greater than5.5 or greater than 7. One capsule from each formulation was placed in aplastic tube containing 50 ml of buffer saline solution adjusted toeither pH 5.7 or pH 7.2. Uncoated gelatin capsules containing the samequantity of SP-304 were used as controls. The plastic tubes wereincubated on a rotatory shaker in a 37° C. incubator. Samples (0.5 ml)were withdrawn from the tube at the indicated time intervals andimmediately used for the bioassay using T84 cells to determine therelease of SP-304 in the solution. The capsule coated with EUDRAGIT®polymer L 30 D-55 (for release at pH greater than 5.5) was incubated atpH 2.5 for 60 min to mimic exposure to stomach acidity. The same capsulewas removed and placed in buffer saline solution (pH 5.7) and sampleswere withdrawn at different times for bioassay. Similarly, the capsulecoated with EUDRAGIT® polymer FSD (for release at pH greater than 7) wassequentially incubated at pH 2.5 (60 min), pH 5.5 (60 min) and then atpH 7.0 for the indicated time interval and samples were withdrawn forbioassay.

FIG. 22: Formulation to deliver SP-304 at pH greater than 7.0 reducedincidence of diarrhea in monkeys. Gelatin capsules were filled withSP-304 to produce a dose of 10 mg/kg body weight. Capsules were coatedwith EUDRAGIT® polymer FSD. Uncoated capsules were used in the controlgroup of monkeys. Effect of treatment was assessed on stool consistency.Results are expressed as cumulative scores of the number of diarrheaincidences in the seven days of treatment period.

FIGS. 23A-B: Formulation to deliver SP-333 at pH greater than 7.0reduced incidence of diarrhea in monkeys. Gelatin capsules were filledwith SP-333 to produce a dose of 10 mg/kg body weight. Capsules werecoated with EUDRAGIT® polymer FSD. Uncoated capsules were used in thecontrol group of monkeys. Effect of treatment was assessed on stoolconsistency. Results are expressed as cumulative scores of the number ofdiarrhea incidences in the seven days of treatment period.

DETAILED DESCRIPTION

The present invention provides novel formulations of GCC agonists whichtarget the release of the GCC agonist to a specific region of thegastrointestinal tract, namely either to the proximal small intestines,preferably the duodenum or jejunum, or to the distal small intestines orlarge intestines, preferably the ileum, terminal ileum, or ascendingcolon. The targeted release GCC agonist formulations of the inventionare useful for the treatment or prevention of gastrointestinal diseasesand disorders. In particular, the formulations which target to theduodenum or jejunum are useful for the treatment or prevention of one ormore of the following: irritable bowel syndrome (preferably constipationpredominant), non-ulcer dyspepsia, chronic intestinalpseudo-obstruction, functional dyspepsia, colonic pseudo-obstruction,duodenogastric reflux, gastro esophageal reflux disease, chronicidiopathic constipation, gastroparesis, heartburn, gastric cancer, andH. pylori infection. Likewise, the formulations which target to theileum, terminal ileum, or ascending colon, are useful for the treatmentor prevention of specific diseases and disorders, including one or moreof the following: ileitis (e.g., post-operative ileitis), Crohn'sdisease, ulcerative colitis, terminal ileitis, and colon cancer.

The targeted release GCC formulations of the invention advantageouslydeliver the GCC agonist to the region of the gastrointestinal tractwhere it will have its greatest therapeutic effect. In a specificembodiment, the GCC agonist is formulated for specific delivery to thedistal small intestines or to the large intestine, preferably the ileum,terminal ileum, or ascending colon. This formulation is particularlyuseful for the treatment of indications such as ulcerative colitis, inwhich the use of convential oral formulations of GCC agonists is limitedby the tendency to produce diarrhea. This adverse reaction is mitigatedor eliminated by the targeted release formulations of the invention,particularly those that target release of the GCC agonist to the ileum,terminal ileum, or ascending colon.

Crohn's disease (CD) and ulcerative colitis (UC) are the principlesyndromes encompassed by the classification of inflammatory boweldisease (IBD). While CD can affect any part of the gastrointestinaltract, it most commonly occurs in the distal ileum and colon, whereas UCby definition affects only the colon. In order to exert theiranti-inflammatory therapeutic effects, orally administered GCC agonistsmust reach the inflamed sites in the distal ileum and colon. Inaccordance with one aspect of the invention, the GCC agonist isformulated for drug release at pH greater than 5.5 for the treatment ofUC and CD. In one embodiment, the GCC agonist is formulated for targeteddelivery to the ileum, preferably using a pH dependent releaseformulation for release at a pH above 5.5. pH dependent releaseformulations are described in more detail, infra, in Section 1.1.“Treatment” in this context refers to the effective induction andmaintenance of remission. Thus, in one embodiment, the inventionprovides a method for the treatment of CD or UC which comprisesadministering to a subject in need thereof a therapeutically effectiveamount of a GCC agonist formulated for release at a pH above 5.5. It hasrecently been shown that gut pH in UC patients is generally more acidicas compared to healthy volunteers and it has also been postulated thatthese variations may affect pH mediated dissolution delivery of drugtreatment (see Rubin, D. T. et al., Colonic pH differs depending on theactivity of ulcerative colitis (UC): Report of two patients with pHmeasurements over time. Poster presented at the Annual ScientificMeeting of the American College of Gastroenterology, Oct. 23-28, 2009,San Diego, Calif., P1116). Further, the colonic pH rose substantiallybetween active inflammation and subsequent clinical quiescence (seeRubin, D. T. et al., Luminal pH and transit time in patients withquiescent ulcerative colitis (US) resembles that of healthy controls.Poster presented at the Annual Scientific Meeting of the AmericanCollege of Gastroenterology, Oct. 23-28, 2009, San Diego, Calif.,P1114). Particularly in UC, some patients fail to achieve remission withstandard outpatient therapy and this failure could possibly be due tothe physiologic differences in lumenal transit time and pH. Thus, afurther advantage of the present invention is to provide GCC agonistformulations specifically designed to deliver bioactive GCC agonists tothe distal ileum or colon of affected patients by utilizing a pHdependent release formulation.

Another advantageous property of the targeted release formulations ofthe invention is that a lower effective dose of the GCC agonist isrequired to achieve the same therapeutic benefit as a GCC agonist whoserelease is not targeted. The targeted release of the GCC agonist furtherensures that the agonist concentration is highest at its site of action,thus reducing the side effects that may be associated with oraladministration.

In one embodiment, the GCC agonist formulation is an oral formulationoptimized for delivery of a GCC agonist to the duodenum or jejunum. In aspecific embodiment, this formulation comprises a core, which containsthe GCC agonist, surrounded by one or more layers of a targetingmaterial. The targeting material is chosen to provide targeted releaseof the GCC agonist to the duodenum or jejunum. In one embodiment, theformulation comprises an outer layer of targeting material whichcomprises a pH dependent polymer that is stable in the low pHenvironment of the stomach (pH 1-2) and begins to disintegrate in a pHrange of from 4.5 to 5.5. This formulation protects the GCC agonistsfrom the acidic environment of the stomach.

In another embodiment, the GCC agonist formulation is optimized fordelivery of a GCC agonist to the ileum, terminal ileum, or ascendingcolon. In a specific embodiment, this formulation comprises a core,which contains the GCC agonist, surrounded by one or more layers of atargeting material. The targeting material is chosen to provide targetedrelease of the GCC agonist to the ileum, terminal ileum, or ascendingcolon. In one embodiment, the formulation comprises three layers oftargeting material: (1) an outer layer which comprises a pH dependentpolymer that is stable in the low pH environment of the stomach (pH 1-2)and begins to disintegrate in a pH range of from 6.5 to 7.5; (2) amiddle layer which comprises a swellable polymer; and (3) an inner layerwhich comprises a pH dependent polymer that begins to disintegrate in apH range of from 6.5 to 7.5. In a preferred embodiment, the pH dependentpolymer is selected from among the EUDRAGIT® polymers, for example,EUDRATGIT® L, S, FS, and E polymers. In one embodiment, the swellablepolymer is hydroxypropylmethylcellulose.

While any GCC agonist known in the art can be formulated according tothe present invention, analogs of uroguanylin and bacterial ST peptidesare preferred. In certain embodiments, the uroguanylin and bacterial STpeptide analogs have superior properties compared to naturallyoccurring, or “wild-type” peptides. For example, the uroguanylin andbacterial ST peptides for use in the present invention are preferablymodified to increase their resistance to degradation at the N-terminusand C-terminus from carboxypeptidases, aminopeptidases, and/or by otherproteolytic enzymes present in the stimulated human intestinal juicesand human gastric juices. In certain embodiments, the GCC agonistformulation comprises a peptide consisting essentially of an amino acidsequence selected from SEQ ID NOs: 1-249. In a preferred embodiment, thepeptide consists essentially of an amino acid sequence selected from SEQID NOs: 1, 8, 9, 55 and 56. The term “consists essentially of” refers toa peptide that is identical to the reference peptide in its amino acidsequence or to a peptide that does not differ substantially in terms ofeither structure or function from the reference peptide. A peptidediffers substantially from the reference peptide if its primary aminoacid sequence varies by more than three amino acids from the referencepeptide or if its activation of cellular cGMP production is reduced bymore than 50% compared to the reference peptide. Preferably,substantially similar peptides differ by no more than two amino acidsand not by more than about 25% with respect to activating cGMPproduction. In preferred embodiments, the GCC agonist is a peptidecomprising at least 12 amino acid residues, and most preferablycomprising between 12 and 26 amino acids. Non-limiting examples of suchanalogs of uroguanylin and bacterial ST peptides are described inSection 1.1 below.

The invention provides methods for treating or preventinggastrointestinal disorders and increasing gastrointestinal motility in asubject in need thereof by administering an effective amount of a GCCagonist formulation to the subject. The term “treating” as used hereinrefers to a reduction, a partial improvement, amelioration, or amitigation of at least one clinical symptom associated with thegastrointestinal disorders being treated. The term “preventing” refersto an inhibition or delay in the onset or progression of at least oneclinical symptom associated with the gastrointestinal disorders to beprevented. The term “effective amount” as used herein refers to anamount that provides some improvement or benefit to the subject. Incertain embodiments, an effective amount is an amount that provides somealleviation, mitigation, and/or decrease in at least one clinicalsymptom of the gastrointestinal disorder to be treated. In otherembodiments, the effective amount is the amount that provides someinhibition or delay in the onset or progression of at least one clinicalsymptom associated with the gastrointestinal disorder to be prevented.The therapeutic effects need not be complete or curative, as long assome benefit is provided to the subject. The term “subject” preferablyrefers to a human subject but may also refer to a non-human primate orother mammal preferably selected from among a mouse, a rat, a dog, acat, a cow, a horse, or a pig.

The gastointestinal disorders that can be treated or prevented accordingto the methods of the invention include, for example, irritable bowelsyndrome (IBS), non-ulcer dyspepsia, chronic intestinalpseudo-obstruction, functional dyspepsia, colonic pseudo-obstruction,duodenogastric reflux, gastroesophageal reflux disease (GERD), ileusinflammation (e.g., post-operative ileus), gastroparesis, heartburn(high acidity in the GI tract), constipation (e.g., constipationassociated with use of medications such as opioids, osteoarthritisdrugs, and osteoporosis drugs), post surgical constipation, andconstipation associated with neuropathic disorders.

In one embodiment, the GCC agonist formulation is optimized for deliveryof a GCC agonist to the duodenum or jejunum. In accordance with thisembodiment, the gastointestinal disorders that can be treated orprevented according to the methods of the invention are selected fromthe group consisting of irritable bowel syndrome (preferablyconstipation predominant), non-ulcer dyspepsia, chronic intestinalpseudo-obstruction, functional dyspepsia, colonic pseudo-obstruction,duodenogastric reflux, gastro esophageal reflux disease, chronicidiopathic constipation, gastroparesis, heartburn, gastric cancer, andH. pylori infection.

In another embodiment, the GCC agonist formulation is optimized fordelivery of a GCC agonist to the ileum, terminal ileum, or ascendingcolon. In accordance with this embodiment, the gastointestinal disordersthat can be treated or prevented according to the methods of theinvention are selected from the group consisting of ileitis (e.g.,post-operative ileitis), Crohn's disease, ulcerative colitis, terminalileitis, and colon cancer.

The invention also provides methods for treating gastrointestinal cancerin a subject in need thereof by administering an effective amount of aGCC agonist formulation to the subject. The term “cancer” in thiscontext includes tissue and organ carcinogenesis including metatases. Inspecific embodiments, the invention provides methods for treating agastrointestinal cancer selected from among gastric cancer, esophagealcancer, pancreatic cancer, colorectal cancer, intestinal cancer, analcancer, liver cancer, gallbladder cancer, or colon cancer.

In accordance with the methods of the present invention, the GCC agonistformulation can be administered alone or in combination with one or moreadditional therapeutic agents to prevent or treat inflammation, cancerand other disorders, particularly of the gastrointestinal tract. In oneembodiment, the GCC agonist formulation is administered in combinationwith one or more additional therapeutic agents selected from the groupconsisting of phosphodiesterase inhibitors, cyclic nucleotides (such ascGMP and cAMP), a laxative (such as SENNA, METAMUCIL, MIRALAX, PEG, orcalcium polycarbophil), a stool softner, an anti-tumor necrosis factoralpha therapy for IBD (such as REMICADE, ENBREL, or HUMAIRA), andanti-inflammatory drugs (such as COX-2 inhibitors, sulfasalazine, 5-ASAderivatives and NSAIDS). In certain embodiments, the GCC agonistformulation is administered in combination with an effective dose of aninhibitor of cGMP-specific phosphodiesterase (cGMP-PDE) eitherconcurrently or sequentially with said GCC agonist. cGMP-PDE inhibitorsinclude, for example, suldinac sulfone, zaprinast, motapizone,vardenifil, and sildenafil. In another embodiment, the GCC agonistformulation is administered in combination with inhibitors of cyclicnucleotide transporters.

1.1 Formulations

The formulations of the invention are optimized for delivery of a GCCagonist to a specific region of the gastrointestinal tract. In apreferred embodiment, the formulations are oral formulations. Theformulations of the invention comprise a core, which contains the GCCagonist, and one or more targeting materials which may form one or morelayers around the core, or which may be formed in a matrix with thecore. The targeting material is chosen to target the release of the GCCagonist to a specific region of the gastrointestinal tract. Thetargeting material preferably comprises one of the following: (1) a pHdependent polymer; (2) a swellable polymer; or (3) a degradablecomposition. Targeting materials are further described in Sections 1.1.1to 1.1.3 below.

In one embodiment, the targeting material is chosen to provide for thepH-dependent release of the GCC agonist. In a preferred embodiment, thetargeting material for a pH-dependent release formulation comprises apH-dependent polymer that is stable in the low pH environment of thestomach (i.e., at pH 1-2) and begins to disintegrate at the pH of theproximal small intestine (pH 4.5-6) or distal ileum (pH 7-8).Preferably, the pH-dependent polymer is a methacrylic acid copolymerselected from among the EUDRAGIT® polymers, which are further describedbelow in Section 1.1.1.

In one embodiment, the targeting material is chosen to provide for acontrolled (time-dependent) release of the GCC agonist. In a preferredembodiment, the targeting material for a controlled release formulationcomprises at least one swellable polymer, as further described below inSection 1.1.2.

In another embodiment, the targeting material for a controlled releaseformulation comprises a degradable compostion such as a natural orsynthetic polymer which is susceptibile to being degraded by at leastone colonic bacterial enzyme. Preferably, the GCC agonist is embedded inthe polymer matrix.

In another embodiment, the targeting material for a controlled releaseformulation comprises a degradable compostion in the form of a carriermolecule covalently conjugated to the GCC agonist by a labile bond whichis stable in the stomach and small intestine but which degrades in thelower gastrointestinal tract, especially the colon, thereby providingfor the targeted release of the GCC agonist. A GCC agonist covalentlyconjugated to a carrier in this manner is referred to herein as a GCC“prodrug.” The formulations comprising a GCC prodrug are furtherdescribed below in Section 1.1.3.

In certain embodiments, the formulation is optimized for delivery of aGCC agonist to the duodenum or jejunum. In one embodiment, theformulation optimized for delivery of a GCC agonist to the duodenum orjejunum comprises a core, which contains the GCC agonist, surrounded bya targeting layer comprised of a pH dependent polymer which degrades ina pH range of 4.5 to 5.5. Preferably the pH dependent polymer is amethacrylic acid copolymer selected from among the EUDRAGIT® polymers.

In certain embodiments, the formulation is optimized for delivery of aGCC agonist to the ileum, terminal ileum, or ascending colon. In oneembodiment, the formulation optimized for delivery of a GCC agonist tothe ileum, terminal ileum, or ascending colon comprises a core, whichcontains the GCC agonist, surrounded by one, two, three, or more layersof targeting materials. At least one targeting material comprises a pHdependent polymer which degrades in a pH range of 6.5 to 7.5. Preferablythe pH dependent polymer is a methacrylic acid copolymer selected fromamong the EUDRAGIT® polymers. Where there is more than one layer oftargeting materials surrounding the core, at least one layer comprises aswellable polymer. Preferably, the swellable polymer is an acryliccopolymer, polyvinylacetate, or a cellulose derivative. In oneembodiment, the swellable polymer is an acrylic copolymer selected fromEUDRAGIT® RL, EUDRAGIT® RS, and EUDRAGIT® NE. In another embodiment, theswellable polymer is a cellulose derivative selected fromethylcellulose, cellulose acetate, hydroxypropylcellulose,hydroxypropylmethylcellulose, hydroxyethylcellulose, methylcellulose,carboxymethylcellulose, and metal salts of carboxymethylcellulose. Inanother embodiment, the formulation comprises EUDRACOL®, which combinesboth pH- and time-dependent EUDRAGIT® polymers. EUDRAGIT® polymers aredescribed below in Sections 1.1.1 and 1.1.2. Further examples ofswellable polymers are described below in Section 1.1.2.

In accordance with the invention, the enteric coating chosen for theformulation is any coating which will achieve the targeting objective ofthe formulation. Examples of suitable enteric coatings include, but arenot limited to, the following: (1) acrylic polymers (anionic polymers ofmethacrylic acid and methacrylates polymers with methacrylic acid as afunctional group) such as the EUDRAGIT® (Degussa) polymers, e.g., forrelease in the duodenum (dissolution above pH 5.5), EUDRAGIT® L 100-55and EUDRAGIT® L 30 D-55; for release in the jejunum (dissolution abovepH 6.0), EUDRAGIT® L 100; for release in the ileum (dissolution above pH7), EUDRAGIT S 100 and EUDRAGIT® FS 30, and COLORCON ACRYL-EZE®; (2)polyvinyl Acetate Phthalate (PVAP) including the COLORCON SURETERIC®Aqueous Enteric Coating System, and the COLORCON OPADRY® Enteric CoatingSystem; (3) hypromellose Phthalate, NF (Hydroxy Propyl Methyl CellulosePhthtalate; HPMCP; HP-55 Shin-Etsu); (4) cellulose acetate phthalate(CAP), such as AQUACOAT CPD®; and (5) cellulose acetate trimellitate(CAT). Further examples of suitable enteric coatings include, withoutlimitation, sustained release blends such as EUDRACOL®, EUDRAPULSE®, andEUDRAMODE®, as well as sustained release polymers such as the EUDRAGIT®RL, RS, and NE polymers.

The GCC agonist-containing core of a formulation of the invention can bein the form of a tablet, a capsule, granules, pellets, or crystals. Incertain embodiments, the core comprises microparticles or microspheres.In one embodiment, the core comprises a cellulose acetate butyratemicrosphere. In some embodiments, the core is coated with one or morelayers of targeting materials. In other embodiments, the core isformulated in a matrix with a targeting material. In certainembodiments, the core matrix is coated with at least one additionaltargeting material.

The GCC-agonist containing core of the present formulations is formedaccording to art-recognized methods. In one embodiment, the core isformed with a pellet-forming agent such as microcrystalline cellulose,low-substituted hydroxypropylcellulose, chitin, chitosan, or anycombination or mixture thereof. Generally, an amount of pellet-formingagent that is less than 20% by weight results in poor sphericity andbroad particle size distribution. Accordingly, the pellet-forming agentof the present formulations is preferably at least 20% by weight. Incertain embodiments, the pellet-forming agent is present at 20% to 95%or 50% to 90% by weight.

The GCC agonist formulation may further comprise one or morepharmaceutically acceptable excipients. The excipients may comprise partof the core or part of one or more outer layers surrounding the core.Preferably, the excipients are present in an amount of 2 to 70% or 5 to50% by weight. The term excipient broadly refers to a biologicallyinactive substance used in combination with the active agents of theformulation. An excipient can be used, for example, as a solubilizingagent, a stabilizing agent, a diluent, an inert carrier, a preservative,a binder, a disintegrant, a coating agent, a flavoring agent, or acoloring agent. Preferably, at least one excipient is chosen to provideone or more beneficial physical properties to the formulation, such asincreased stability and/or solubility of the active agent(s).

A “pharmaceutically acceptable” excipient is one that has been approvedby a state or federal regulatory agency for use in animals, andpreferably for use in humans, or is listed in the U.S. Pharmacopia, theEuropean Pharmacopia or another generally recognized pharmacopia for usein animals, and preferably for use in humans. Examples of excipientsinclude certain inert proteins such as albumins; hydrophilic polymerssuch as polyvinylpyrrolidone; amino acids such as aspartic acid (whichmay alternatively be referred to as aspartate), glutamic acid (which mayalternatively be referred to as glutamate), lysine, arginine, glycine,and histidine; fatty acids and phospholipids such as alkyl sulfonatesand caprylate; surfactants such as sodium dodecyl sulphate andpolysorbate; nonionic surfactants such as such as TWEEN®, PLUIRONICS®,or polyethylene glycol (PEG); carbohydrates such as glucose, sucrose,mannose, maltose, trehalose, and dextrins, including cyclodextrins;polyols such as sorbitol; chelating agents such as EDTA; andsalt-forming counter-ions such as sodium. Particularly preferred arehydrophilic excipients which reduce the protein binding activity andaggregation of GCC agionist peptides.

In some embodiments, the GCC agonist formulation further comprises oneor more excipients selected from among an absorption enhancer, a binder,a disintegrant, and a hardness enhancing agent. In other embodiments,the formulation further comprises one or more excipients selected fromamong a wicking agent, a stabilizer, a flow regulating agent, alubricant, an antioxidant, a chelating agent, or a sequestrate.

Non-limiting examples of suitable binders include starch,polyvinylpyrrolidone (POVIDONE), low molecular weighthydroxypropylcellulose, low molecular weighthydroxypropylmethylcellulose, low molecular weightcarboxymethylcellulose, ethylcellulose, gelatin, polyethylene oxide,acacia, dextrin, magnesium aluminum silicate, and polymethacrylates.Non-limiting examples of a disintegrant include croscarmellose sodiumcrospovidone (cross-linked PVP), sodium carboxymethyl starch (sodiumstarch glycolate), pregelatinized starch (starch 1500), microcrystallinestarch, water insoluble starch, calcium carboxymethyl cellulose, andmagnesium aluminum silicate (Veegum). In certain embodiments, a binderis selected from polyvinylpyrrolidone and sodium carboxymethylcellulose.

Non-limiting examples of a wicking agent include colloidal silicondioxide, kaolin, titanium oxide, fumed silicon dioxide, alumina,niacinamide, sodium lauryl sulfate, low molecular weight polyvinylpyrrolidone, m-pyrol, bentonite, magnesium aluminum silicate, polyester,polyethylene, and mixtures thereof. In certain embodiments, a wickingagent is selected from sodium lauryl sulfate, colloidal silicon dioxide,and low molecular weight polyvinyl pyrrolidone.

Non-limiting examples of a stabilizer include butyl hydroxyanisole,ascorbic acid, citric acid, and mixtures thereof. Preferably, thestabilizer is a basic substance which can elevate the pH of an aqueoussolution or dispersion of the formulation to at least about pH 6.8.Examples of such basic substances include, for example, antacids such asmagnesium aluminometasilicate, magnesium aluminosilicate, magnesiumaluminate, dried aluminum hydroxide, synthetic hydrotalcite, syntheticaluminum silicate, magnesium carbonate, precipitated calcium carbonate,magnesium oxide, aluminum hydroxide, and sodium hydrogencarbonate. Otherexamples incldue pH-regulating agents such as L-arginine, sodiumphosphate, disodium hydrogen phosphate, sodium dihydrogenphosphate,potassium phosphate, dipotassium hydrogenphosphate, potassiumdihydrogenphosphate, disodium citrate, sodium succinate, ammoniumchloride, and sodium benzoate. In certain embodiments, a stabilizer isselected from ascorbic acid and magnesium aluminometasilicate.

In an embodiment where the stabilizer is a basic substance, the basicsubstance can be an inorganic water-soluble compound or a inorganicwater-insoluble compound. Non-limiting examples of an inorganicwater-soluble compounds for use as a stabilizer include carbonate saltssuch as sodium carbonate, potassium carbonate, sodium bicarbonate, orpotassium hydrogen carbonate; phosphate salts such as anhydrous sodiumphosphate, potassium phosphate, calcium dibasic phosphate, or trisodiumphosphate; and alkali metal hydroxides, such as sodium, potassium, orlithium hydroxide. Non-limiting examples of inorganic water-insolublecompounds for use as a stabilizer include suitable alkaline compoundscapable of imparting the requisite basicity, such as those commonlyemployed in antiacid compositions, for example, magnesium oxide,magnesium hydroxide, magnesium carbonate, magnesium hydrogen carbonate,aluminum hydroxide, calcium hydroxide, or calcium carbonate; compositealuminum-magnesium compounds, such as magnesium aluminum hydroxide;silicate compounds such as magnesium aluminum silicate (Veegum F),magnesium aluminometasilicate (Nesulin FH2), magnesium aluminosilicate(Nisulin A); and pharmaceutically acceptable salts of phosphoric acidsuch as tribasic calcium phosphate.

Non-limiting examples of a flow regulating agents include a colloidalsilicon dioxide and aluminum silicate.

Non-limiting examples of a lubricant include stearate salts, such asmagnesium stearate, calcium stearate, and sodium stearate, stearic acid,talc, sodium stearyl fumarate, sodium lauryl sulfate, sodium benzoate,polyethylene glycol, polyvinyl alcohol, glycerol behenate compritol(glycerol behenate), corola oil, glyceryl palmitostearate, hydrogenatedvegetable oil, magnesium oxide, mineral oil, poloxamer, and combinationsthereof. In certain embodiments, a lubricant is selected from talc andmagnesium stearate.

Non-limiting examples of antioxidants include 4,4 (2,3 dimethyltetramethylene dipyrochatechol), tocopherol-rich extract (naturalvitamin E), α-tocopherol (synthetic Vitamin E), β-tocopherol,γ-tocopherol, δ-tocopherol, butylhydroxinon, butyl hydroxyanisole (BHA),butyl hydroxytoluene (BHT), propyl gallate, octyl gallate, dodecylgallate, tertiary butylhydroquinone (TBHQ), fumaric acid, malic acid,ascorbic acid (Vitamin C), sodium ascorbate, calcium ascorbate,potassium ascorbate, ascorbyl palmitate, ascorbyl stearate, citric acid,sodium lactate, potassium lactate, calcium lactate, magnesium lactate,anoxomer, erythorbic acid, sodium erythorbate, erythorbin acid, sodiumerythorbin, ethoxyquin, glycine, gum guaiac, sodium citrates (monosodiumcitrate, disodium citrate, trisodium citrate), potassium citrates(monopotassium citrate, tripotassium citrate), lecithin, polyphosphate,tartaric acid, sodium tartrates (monosodium tartrate, disodiumtartrate), potassium tartrates (monopotassium tartrate, dipotassiumtartrate), sodium potassium tartrate, phosphoric acid, sodium phosphates(monosodium phosphate, disodium phosphate, trisodium phosphate),potassium phosphates (monopotassium phosphate, dipotassium phosphate,tripotassium phosphate), calcium disodium ethylene diamine tetra-acetate(Calcium disodium EDTA), lactic acid, trihydroxy butyrophenone andthiodipropionic acid.

In certain embodiments, the core of the formulation comprises anantioxidant and both a chelator and a sequestrate. The chelating agentacts to remove trace quantities of metals which might otherwise bind tothe GCC agonist and cause loss of activity, for example throughoxidation. The sequestrate preferably has several hydroxyl and/orcarboxylic acid groups which provide a supply of hydrogen forregeneration of the inactivated antioxidant free radical. Non-limitingexamples of chelating agents include antioxidants, dipotassium edentate,disodium edentate, edetate calcium disodium, edetic acid, fumaric acid,malic acid, maltol, sodium edentate, and trisodium edetate. Non-limitingexamples of sequestrates include citric acid and ascorbic acid.

In some embodiments, the formulation further comprises a filler.Preferably, the filler is present in an amount of from 10% to 85% byweight. Non-limiting examples of suitable materials for use as a fillerinclude starch, lactitol, lactose, an inorganic calcium salt,microcrystalline cellulose, sucrose, and combinations thereof. In someembodiments, the filler comprises microcrystalline cellulose.Preferably, the microcrystalline cellulose has a particle size of lessthan about 100 microns, and most preferably the microcrystallinecellulose has a particle size of about 50 microns.

In some embodiments, the core optionally includes a buffering agent suchas an inorganic salt compound and an organic alkaline salt compound.Non-limiting examples of a buffering agent include potassiumbicarbonate, potassium citrate, potassium hydroxide, sodium bicarbonate,sodium citrate, sodium hydroxide, calcium carbonate, dibasic sodiumphosphate, monosodium glutamate, tribasic calcium phosphate,monoethanolamine, diethanolamine, triethanolamine, citric acidmonohydrate, lactic acid, propionic acid, tartaric acid, fumaric acid,malic acid, and monobasic sodium phosphate.

In some embodiments, the core further comprises a preservative.Non-limiting examples of a preservative include an antioxidant,dipotassium edentate, disodium edentate, edetate calcium disodium,edetic acid, fumaric acid, malic acid, maltol, sodium edentate, andtrisodium edentate.

The GCC agonist formulations of the invention are preferably optimizedfor oral delivery. However, in some embodiments, the formulations may beprepared in the form of suppositories (e.g., with conventionalsuppository bases such as cocoa butter and other glycerides) orretention enemas for rectal delivery. Solid oral dosage forms mayoptionally be treated with coating systems (e.g. Opadry® fx film coatingsystem, for example Opadry® blue (OY-LS-20921), Opadry® white(YS-2-7063), Opadry® white (YS-1-7040), and black ink (S-1-8 106).

1.1.1 pH Dependent Release Formulations

In certain embodiments, the formulations of the invention comprise apH-dependent targeting material that is pharmacologically inactive,meaning that it is excreted without being absorbed or metabolized. Insome embodiments, the GCC agonist-loaded core is coated with apH-dependent material. In other embodiments, the pH-dependent materialcomprises part of an outer layer which surrounds the core, for examplein certain embodiments of a controlled (time-dependent) releaseformulation. In some embodiments, the GCC agonist-loaded core is formedas a matrix with a pH-dependent material. Preferably, the pH-dependentmaterial comprises a pH-dependent polymer.

Preferably, the pH-dependent polymer is stable in the low pH environmentof the stomach (i.e., at pH 1-2) and begins to disintegrate at thehigher pH of the small intestine (pH 6-7) or distal ileum (pH 7-8). Incertain embodiments, the polymer begins to disintegrate at pH 4.5-4.8,pH 4.8-5.0, pH 5.0-5.2, pH 5.2-5.4, pH 5.4-5.8, pH 5.8-6.0, pH 6.0-6.2,pH 6.2-6.4, pH 6.4-6.6, pH 6.6-6.8, pH 6.8-7.0, pH 7.0-7.2, or pH7.2-7.4. In certain embodiments, the polymer begins to disintegrate atpH 4.5-5.5, pH 5.5-6.5, or pH 6.5-7.5. The pH at which a pH-sensitivepolymer begins to disintegrate is also referred to herein as the“threshold pH” of the polymer.

In certain embodiments, the pH-dependent polymer is a methacrylic acidcopolymer, a polyvinyl acetate phthalate, a hydroxypropylmethylcellulosephthalate, a cellulose acetate trimelliate, a cellulose acetatephthalate, or a hydroxypropyl methyl cellulose acetate succinate.

In a preferred embodiment, the pH-dependent polymer is a methacrylicacid copolymer selected from among the EUDRAGIT® polymers. EUDRAGIT®polymers are available in a wide range of different concentrations andphysical forms, including aqueous solutions, aqueous dispersion, organicsolutions, and solid substances. The pharmaceutical properties of thepolymers are determined by the chemical properties of their functionalgroups. For example, EUDRAGIT® L, S, FS and E polymers have acidic oralkaline groups that are pH-dependent. Enteric EUDRAGIT® coatingsprovide protection against release of the GCC agonist in the stomach andenable controlled release in the intestine. In certain embodiments,anionic EUDRAGIT® grades containing carboxyl groups are mixed with eachother to provide pH-dependent release of the GCC agonist. In certainembodiments, EUDRAGIT® L and S grades are used for enteric coatings. Inone embodiment, EUDRAGIT® FS 30D is used for controlled release in thecolon. The various EUDRAGIT® polymers are further described ininternational pharmacopeias such as Ph.Eur., USP/NF, DMF and JPE.

In specific embodiments, the pH-dependent polymer is a methacrylic acidcopolymer selected from EUDRAGIT® L100, having a threshold pH of 6.0;EUDRAGIT® S100, having a threshold pH of 7.0; EUDRAGIT® L-30D, having athreshold pH of 5.6; EUDRAGIT® FS 30D, having a threshold pH of 6.8; orEUDRAGIT® L100-55, having a threshold pH of 5.5, or a combinationthereof.

1.1.2 Controlled Release Formulations

In one embodiment, the GCC agonist formulation comprises a targetingmaterial which provides a controlled (time-dependent) release of the GCCagonist. Controlled release in this context includes delayed sustainedrelease, delayed controlled release, delayed slow release, delayedprolonged release, delayed extended release, and a sudden release or“burst.”

Preferably, the controlled release formulation comprises a slowlydisintegrating core comprising the GCC agonist surrounded by thetargeting material. The targeting material preferably comprises at leastone swellable polymer. Non-limiting examples of swellable polymers foruse in a controlled release formulation of the invention include acryliccopolymers, e.g., EUDRAGIT® RL, EUDRAGIT® RS, or EUDRAGIT® NE;polyvinylacetate, e.g., KOLLICOAT® SR 30D; and cellulose derivativessuch as ethylcellulose or cellulose acetate, e.g., SURELEASE® andAQUACOAT® ECD. In a preferred embodiment, the targeting materialcomprises one or more of EUDRAGIT® RL, EUDRAGIT® RS, or EUDRAGIT® NE toprovide controlled time release of the GCC agonist by pH-independentswelling. In a particular embodiment, the targeting material comprisesEUDRAGIT® RL:RS (2:8) and an outing coating comprising EUDRAGIT® FS.

Further non-limiting examples of swellable polymers that can be used inthe sustained release formulations of the invention includepoly(hydroxalkyl methacrylate) having a molecular weight of from 30,000to 5,000.000; kappa-carrageenan; polyvinylpyrrolidone having a molecularweight of from 10,000 to 360,000; anionic and cationic hydrogels;polyelectrolyte complexes; poly(vinyl alcohol) having low amounts ofacetate, cross-linked with glyoxal, formaldehyde, or glutaraldehyde andhaving a degree of polymerization from 200 to 30,000; a mixturecomprising methyl cellulose, cross-linked agar and carboxymethylcellulose; a water-insoluble, water-swellable copolymer produced byforming a dispersion of finely divided maleic anhydride with styrene,ethylene, propylene, butylene or isobutylene; water-swellable polymersof N-vinyl lactams; polysaccharide, water swellable gums, high viscosityhydroxylpropylmethyl cellulose and/or mixtures thereof. In certainembodiments, the swellable polymer is selected from the group consistingof calcium pectinate, cross-linked polysaccharide, water insolublestarch, microcrystalline cellulose, water insoluble cross-linkedpeptide, water insoluble cross-linked protein, water insolublecross-linked gelatin, water insoluble cross-linked hydrolyzed gelatin,water insoluble cross-linked collagen, modified cellulose, andcross-linked polyacrylic acid. Non-limiting examples of a cross-linkedpolysaccharide include insoluble metal salts or cross-linked derivativesof alginate, pectin, xantham gum, guar gum, tragacanth gum, and locustbean gum, carrageenan, metal salts thereof, and covalently cross-linkedderivatives thereof. Non-limiting examples of modified cellulose includecross-linked derivatives of hydroxypropylcellulose,hydroxypropylmethylcellulose, hydroxyethylcellulose, methylcellulose,carboxymethylcellulose, and metal salts of carboxymethylcellulose.

In certain embodiments, the swellable core also comprises a wickingagent such as silicon dioxide. The wicking agent may also be selectedfrom a disintegrant such as microcrystalline cellulose to enhance thespeed of water uptake. Other suitable wicking agents include, but arenot limited to, kaolin, titanium dioxide, fumed silicon dioxide,alumina, niacinamide, sodium lauryl sulfate, low molecular weightpolyvinyl pyrrolidone, m-pyrol, bentonite, magnesium aluminum silicate,polyester, polyethylene, and mixtures thereof.

In certain embodiments, the targeting material, which may comprise partof the core and/or form one or more layers coating the core, optionallyfurther comprises at least one of a lubricant, a flow promoting agent, aplasticizer, an anti-sticking agent, surfactant, wetting agent,suspending agent and dispersing agent.

In certain embodiments, the targeting material comprises a waterinsoluble polymer and a pore-forming agent. Non-limiting examples ofpore forming agents include saccharose, sodium chloride, potassiumchloride, polyvinylpyrrolidone, and/or polyethyleneglycol, water solubleorganic acids, sugars and sugar alcohol. In certain embodiments, thepore forming agent forms part of an outer layer or coating. In otherembodiments, the pore forming agent is distributed uniformly throughoutthe water insoluble polymer.

In one embodiment, the targeting material comprises a compressioncoating. Non-limiting examples of materials that can be used as acompression coating include a gum selected from the group consisting ofxanthan gum, locust bean gum, galactans, mannans, alginates, gum karaya,pectin, agar, tragacanth, accacia, carrageenan, tragacanth, chitosan,agar, alginic acid, hydrocolloids acacia catechu, salai guggal, indianbodellum, copaiba gum, asafetida, cambi gum, Enterolobium cyclocarpum,mastic gum, benzoin gum, sandarac, gambier gum, butea frondosa (Flame ofForest Gum), myrrh, konjak mannan, guar gum, welan gum, gellan gum, taragum, locust bean gum, carageenan gum, glucomannan, galactan gum, sodiumalginate, tragacanth, chitosan, xanthan gum, deacetylated xanthan gum,pectin, sodium polypectate, gluten, karaya gum, tamarind gum, ghattigum, Accaroid/Yacca/Red gum, dammar gum, juniper gum, ester gum,ipil-ipil seed gum, gum talha (acacia seyal), and cultured plant cellgums including those of the plants of the genera: acacia, actinidia,aptenia, carbobrotus, chickorium, cucumis, glycine, hibiscus, hordeum,letuca, lycopersicon, malus, medicago, mesembryanthemum, oryza, panicum,phalaris, phleum, poliathus, polycarbophil, sida, solanum, trifolium,trigonella, Afzelia africana seed gum, Treculia africana gum, detariumgum, cassia gum, carob gum, Prosopis africana gum, Colocassia esulentagum, Hakea gibbosa gum, khaya gum, scleroglucan, and zea, as well asmixtures of any of the foregoing.

In some embodiments, the targeting material further comprises aplasticizer, a stiffening agent, a wetting agent, a suspending agent, ora dispersing agent, or a combination thereof. Non-limiting examples of aplasticizer include dibutyl sebacate, polyethylene glycol andpolypropylene glycol, dibutyl phthalate, diethyl phthalate, triethylcitrate, tributyl citrate, acetylated monoglyceride, acetyl tributylcitrate, triacetin, dimethyl phthalate, benzyl benzoate, butyl and/orglycol esters of fatty acids, refined mineral oils, oleic acid, castoroil, corn oil, camphor, glycerol and sorbitol or a combination thereof.In one embodiment, the stiffening agent comprises cetyl alcohol.Non-limiting examples of wetting agents include a poloxamer,polyoxyethylene ethers, polyoxyethylene sorbitan fatty acid esters,polyoxymethylene stearate, sodium lauryl sulfate, sorbitan fatty acidesters, benzalkonium chloride, polyethoxylated castor oil, and docusatesodium. Non-limiting examples of suspending agents include alginic acid,bentonite, carbomer, carboxymethylcellulose, carboxymethylcellulosecalcium, hydroxyethylcellulose, hydroxypropylcellulose, microcrystallinecellulose, colloidal silicon dioxide, dextrin, gelatin, guar gum,xanthan gum, kaolin, magnesium aluminum silicate, maltitol, medium chaintriglycerides, methylcellulose, polyoxyethylene sorbitan fatty acidesters, polyvinylpyrrolidinone, propylene glycol alginate, sodiumalginate, sorbitan fatty acid esters, and tragacanth. Non-limitingexamples of dispersing agents include poloxamer, polyoxyethylenesorbitan fatty acid esters and sorbitan fatty acid esters.

In certain embodiments, the targeted release formulation furthercomprises an outer enteric coating over the targeted release material.Preferably, the enteric coating is selected from the group consisting ofcellulose acetate phthalate, hydroxy propyl methyl cellulose acetatesuccinate, EUDRAGIT® L100 and EUDRAGIT® L30D-55.

1.1.2.1 Burst Formulation

In one embodiment, the GCC agonist formulation is a time-delayedformulation designed to release the GCC agonist in a fast burst in thecolon or small intestine (“burst formulation”). The formulationcomprises a core and an outer layer. The core comprises at least one GCCagonist and at least one burst controlling agent. In certainembodiments, the core further comprises at least one disintegrantselected from the group consisting of croscarmellose sodium,crospovidone (cross-linked PVP), sodium carboxymethyl starch (sodiumstarch glycolate), cross-linked sodium carboxymethyl cellulose(Croscarmellose), pregelatinized starch (starch 1500), microcrystallinestarch, water insoluble starch, calcium carboxymethyl cellulose, andmagnesium aluminum silicate, or a combination thereof. In otherembodiments, the core further comprises at least one of an absorptionenhancer, a binder, a hardness enhancing agent, a buffering agent, afiller, a flow regulating agent, a lubricant, a synergistic agent, achelator, an antioxidant, a stabilizer and a preservative. Optionally,the core also comprises one or more other excipients.

The burst controlling agent in the core preferably comprises a waterinsoluble polymer for controlling the rate of penetration of water intothe core and raising the internal pressure (osmotic pressure) inside thecore. Such a burst controlling agent is preferably able to swell uponcontact with liquid. Non-limiting examples of suitable water insolublepolymers include cross-linked polysaccharide, water insoluble starch,microcrystalline cellulose, water insoluble cross-linked peptide, waterinsoluble cross-linked protein, water insoluble cross-linked gelatin,water insoluble cross-linked hydrolyzed gelatin, water insolublecross-linked collagen modified cellulose, and cross-linked polyacrylicacid. In one embodiment, the water insoluble polymer is a cross-linkedpolysaccharide selected from the group consisting of insoluble metalsalts or cross-linked derivatives of alginate, pectin, xanthan gum, guargum, tragacanth gum, and locust bean gum, carrageenan, metal saltsthereof, and covalently cross-linked derivatives thereof. In oneembodiment, the water insoluble polymer is a modified cellulose selectedfrom the group consisting of cross-linked derivatives ofhydroxypropylcellulose, hydroxypropylmethylcellulose,hydroxyethylcellulose, methylcellulose, carboxymethylcellulose, andmetal salts of carboxymethylcellulose. In another embodiment, the waterinsoluble polymer is selected from calcium pectinate, microcrystallinecellulose, or a combination thereof.

The outer layer comprises a water insoluble hydrophobic carrier and apore forming agent comprised of a water insoluble hydrophilic particularmatter. The pore forming agent is a water permeable agent which allowsentry of liquid into the core. Optionally, the outer layer furthercomprises at least one of a wetting agent, a suspending agent, adispersing agent, a stiffening agent, and a plasticizer.

In certain embodiments, the water insoluble hydrophobic carrier isselected from the group consisting of adimethylaminoethylacrylate/ethylmethacrylate copolymer, the copolymerbeing based on acrylic and methacrylic acid esters with a low content ofquaternary ammonium groups, wherein the molar ratio of the ammoniumgroups to the remaining neutral (meth)acrylic acid esters isapproximately 1:20, the polymer corresponding to USP/NF “AmmonioMethacrylate Copolymer Type A”, anethylmethacrylate/chlorotrimethylammoniumethyl methacrylate copolymer,the copolymer based on acrylic and methacrylic acid esters with a lowcontent of quaternary ammonium groups wherein the molar ratio of theammonium groups to the remaining neutral (meth)acrylic acid esters is1:40, the polymer corresponding to USP/NF “Ammonio MethacrylateCopolymer Type B”, a dimethylaminoethylmethacrylate/methylmethacrylateand butylmethacrylate copolymer, a copolymer based on neutralmethacrylic acid esters and dimethylaminoethyl methacrylate esterswherein the polymer is cationic in the presence of acids, anethylacrylate and methylacrylate/ethylmethacrylate and methylmethylacrylate copolymer, the copolymer being a neutral copolymer basedon neutral methacrylic acid and acrylic acid esters, ethylcellulose,shellac, zein, and waxes.

In certain embodiments, the water insoluble particulate matter is ahydrophilic yet water insoluble polymer, preferably selected from thegroup consisting of a water insoluble cross-linked polysaccharide, awater insoluble cross-linked protein, a water insoluble cross-linkedpeptide, water insoluble cross-linked gelatin, water insolublecross-linked hydrolyzed gelatin, water insoluble cross-linked collagen,water insoluble cross linked polyacrylic acid, water insolublecross-linked cellulose derivatives, water insoluble cross-linkedpolyvinyl pyrrolidone, micro crystalline cellulose, insoluble starch,micro crystalline starch and a combination thereof. Most preferably, thewater insoluble particulate matter is microcrystalline cellulose.

In certain embodiments, the burst formulation further comprises anenteric coating on the outer layer. The enteric coating is preferablyselected from the group consisting of cellulose acetate phthalate,hydroxy propyl methyl cellulose acetate succinate, and a EUDRAGIT®polymer such as EUDRAGIT® L100 or EUDRAGIT® L30D-55.

1.1.3 Biodegradable Formulations

In one embodiment, the GCC agonist formulation comprises a natural orsynthetic polymer which is susceptibile to being degraded by at leastone colonic bacterial enzyme. Preferably, the GCC agonist is embedded inthe polymer matrix. Non-limiting examples of such polymers includepolymers of polysaccharides such as amylase, chitosan, chondroitinsulfate, cyclodextrin, dextran, guar gum, pectin, and xylan. Preferably,the natural or synthetic polymer is gelled or crosslinked with a cationsuch as a zinc cation, for example from zinc sulfate, zinc chloride, orzinc acetate. The formulation is preferably in the form of ionicallycrosslinked beads which are subsequently coated with an enteric coating.The enteric coating can comprise any suitable enteric coating material,such as hydroxypropylmethyl cellulose phthalate, polyvinyl acetatephthalate, cellulose acetate phthalate, hydroxypropylmethyl celluloseacetate succinate, alginic acid, and sodium alginate, or a EUDRAGIT®polymer.

In another embodiment, the GCC agonist formulation comprises a GCCagonist covalently conjugated to a carrier molecule such that thecovalent bond between the GCC agonist and the carrier is stable in thestomach and small intestine but labile in the lower gastrointestinaltract, especially the colon. The GCC agonist covalently linked to acarrier molecule is referred to as the “GCC prodrug.” In certainembodiments, the GCC prodrug comprises a GCC agonist covalentlyconjugated to a carrier molecule via an azo bond or a glycosidic bond.In other embodiments, the GCC prodrug comprises a glucuronide, acyclodextrin, a dextran ester, or a polar amino acid. In certainembodiments, the GCC prodrug is a polymeric prodrug. In one embodiment,the polymeric prodrug comprises polyamides containing azo groups.

1.2 GCC Agonists

The GCC agonists for use in the formulations and methods of theinvention bind to guanylate cyclase C and stimulate intracellularproduction of cGMP. Optionally, the GCC agonists induce apoptosis andinhibit proliferation of epithelial cells. The term, “guanylate cyclaseC” refers to a transmembrane form of guanylate cyclase that acts as theintestinal receptor for the heat-stable toxin (ST) peptides secreted byenteric bacteria. Guanylate cyclase C is also the receptor for thenaturally occurring peptides guanylin and uroguanylin. The possibilitythat there may be different receptors for each of these peptides has notbeen excluded. Hence, the term “guanylate cyclase C” may also encompassa class of transmembrane guanylate cyclase receptors expressed onepithelial cells lining the gastrointestinal mucosa.

The term “GCC agonist” refers to both peptides and non-peptide compoundssuch as that bind to an intestinal guanylate cyclase C and stimulate theintracellular production of cGMP. Where the GCC agonist is a peptide,the term encompasses biologically active fragments of such peptides andpro-peptides that bind to guanylate cyclase C and stimulate theintracellular production of cGMP.

Preferably, the GCC agonists for use in the formulations and methods ofthe invention stimulate intracellular cGMP production at higher levelsthan naturally occurring GCC agonists such as uroguanylin, guanylin, andST peptides. In some embodiments, the GCC agonists stimulateintracellular cGMP production at higher levels than the peptidedesignated SP-304 (SEQ ID NO:1). In specific embodiments, a GCC agonistfor use in the formulations and methods of the invention stimulates 5%,10%, 20%, 30%, 40%, 50%, 75%, 90% or more intracellular cGMP compared touroguanylin, guanylin, lymphoguanylin, linaclotide, ST peptides, orSP-304. The terms “induce” and “stimulate” are used interchangeablythroughout the specification.

Preferably, the GCC agonists for use in the formulations and methods ofthe invention are more stable than naturally occurring GCC agonists suchas uroguanylin, guanylin, and ST peptides. In some embodiments, the GCCagonists are more stable than the peptide designated SP-304. “Stability”in this context refers to resistance to degradation in gastrointestinalfluid and/or intestinal fluid (or simulated gastrointestinal orintestinal fluids) compared to the reference peptide. For example, theGCC agonists for use in the formulations and methods of the inventionpreferably degrade 2%, 3%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 75%, 90% orless compared to naturally occurring GCC angonists and/or SP-304.

The GCC agonists for use in the formulations and methods of theinvention are preferably peptides. In some embodiments, the GCC agonistpeptide is less than 30 amino acids in length. In particularembodiments, the GCC agonist peptide is less than or equal to 30, 25,20, 15, 14, 13, 12, 11, 10, or 5 amino acids in length. Examples of GCCagonist peptides for use in the formulations and methods of theinvention include those described in U.S. Ser. No. 12/133,344, filedJun. 4, 2008, Ser. No. 12/478,505, filed Jun. 4, 2009; Ser. No.12/478,511, filed Jun. 4, 2009; Ser. No. 12/504,288, filed Jul. 16,2009; and U.S. Provisional Application Ser. No. 60/933,194, filed Jun.4, 2007; 61/058,888, filed Jun. 4, 2008; 61/058,892, filed Jun. 4, 2008;and 61/081,289, filed Jul. 16, 2008, each of which is incorporated byreference herein in its entirety.

Specific examples of GCC agonist peptides for use in the formulationsand methods of the invention include those described in Tables I-VIIbelow. As used Tables I-VII, the terms “PEG3” or “3PEG” refer to apolyethylene glycol such as aminoethyloxy-ethyloxy-acetic acid (AeeA),and polymers thereof. The term “X_(aa)” refers to any natural orunnatural amino acid or amino acid analogue. The term “M_(aa)” refers toa cysteine (Cys), penicillamine (Pen) homocysteine, or3-mercaptoproline. The term “Xaa_(n1)” is meant to denote an amino acidsequence of any natural or unnatural amino acid or amino acid analoguethat is one, two or three residues in length; Xaa_(n2) is meant todenote an amino acid sequence that is zero or one residue in length; andXaa_(n3) is meant to denote an amino acid sequence zero, one, two,three, four, five or six residues in length. Additionally, any aminoacid represented by Xaa, Xaa_(n1), Xaa_(n2), or Xaa_(n3) may be anL-amino acid, a D-amino acid, a methylated amino acid or any combinationof thereof. Optionally, any GCC agonist peptide represented by FormulasI to XX in the tables may contain on or more polyethylene glycolresidues at the the N-terminus, C-terminus or both.

In certain embodiments, a GCC agonist formulation of the inventioncomprises a peptide selected from SEQ ID NOs: 1-249, the sequences ofwhich are set forth below in Tables I to VII below. In one embodiment, aGCC agonist formulation comprises the peptide designated by SEQ IDNOs:1, 8, 9, 55, or 56.

In certain embodiments, a GCC agonist formulation of the inventioncomprises a peptide that is substantially equivalent to a peptideselected from SEQ ID NOs: 1-249. The term “substantially equivalent”refers to a peptide that has an amino acid sequence equivalent to thatof the binding domain where certain residues may be deleted or replacedwith other amino acids without impairing the peptide's ability to bindto an intestinal guanylate cyclase receptor and stimulate fluid andelectrolyte transport.

1.2.1 GCC Agonist Peptides

In a preferred embodiment, the GCC agonists for use in the formulationsand methods of the invention are GCC agonist peptides. In certainembodiments, the GCC agonist peptides are analogues of uroguanylin or abacterial ST peptide. Uroguanylin is a circulating peptide hormone withnatriuretic activity. An ST peptide is a member of a family of heatstable enterotoxins (ST peptides) secreted by pathogenic strains of E.coli and other enteric bacteria that activate guanylate cyclase receptorand cause secretory diarrhea. Unlike bacterial ST peptides, the bindingof uroguanylin to guanylate cyclase receptor is dependent on thephysiological pH of the gut. Therefore, uroguanylin is expected toregulate fluid and electrolyte transport in a pH dependent manner andwithout causing severe diarrhea.

The GCC agonist peptides for use in the formulations and methods of theinvention can be polymers of L-amino acids, D-amino acids, or acombination of both. For example, in various embodiments, the peptidesare D retro-inverso peptides. The term “retro-inverso isomer” refers toan isomer of a linear peptide in which the direction of the sequence isreversed and the chirality of each amino acid residue is inverted. See,e.g., Jameson et al., Nature, 368, 744-746 (1994); Brady et al., Nature,368, 692-693 (1994). The net result of combining D-enantiomers andreverse synthesis is that the positions of carbonyl and amino groups ineach amide bond are exchanged, while the position of the side-chaingroups at each alpha carbon is preserved. Unless specifically statedotherwise, it is presumed that any given L-amino acid sequence of theinvention may be made into an D retro-inverso peptide by synthesizing areverse of the sequence for the corresponding native L-amino acidsequence.

The GCC agonist peptides for use in the formulations and methods of theinvention are able to induce intracellular cGMP production in cells andtissues expressing guanylate cyclase C. In certain embodiments, the GCCagonist peptide stimulates 5%, 10%, 20%, 30%, 40%, 50%, 75%, 90% or moreintracellular cGMP compared to naturally occurring GCC agonists such asuroguanylin, guanylin, or ST peptides. Optionally, the GCC agonistpeptide stimulates 5%, 10%, 20%, 30%, 40%, 50%, 75%, 90% or moreintracellular cGMP compared SP-304 (SEQ ID NO:1). In furtherembodiments, the GCC agonist peptide stimulates apoptosis, e.g.,programmed cell death, or activate the cystic fibrosis transmembraneconductance regulator (CFTR).

In some embodiments, the GCC agonist peptides for use in theformulations and methods of the invention are more stable than naturallyoccurring GCC agonists and/or SP-304 (SEQ ID NO:1), SP-339 (linaclotide)(SEQ ID NO: 55) or SP-340 (SEQ ID NO: 56). For example, the GCC agonistpeptide degrades 2%, 3%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 75%, 90% orless compared to naturally occurring GCC agonists and/or SP-304, SP-339(linaclotide) or SP-340. In certain embodiments, the GCC agonistpeptides for use in the formulations and methods of the invention aremore stable to proteolytic digestion than naturally occurring GCCagonists and/or SP-304 (SEQ ID NO:1), SP-339 (linaclotide) (SEQ ID NO:55) or SP-340 (SEQ ID NO: 56). In one embodiment, a GCC agonist peptideis pegylated in order to render the peptides more resistant towardsprotealysis by enzymes of the gastrointestinal tract. In a preferredembodiment, the GCC agonist peptide is pegylated with theaminoethyloxy-ethyloxy-acetic acid (Aeea) group at its C-terminal end,at its N-terminal end, or at both termini.

Specific examples of GCC agonist peptides that can be used in themethods and formulations of the invention include a peptide selectedfrom the group designated by SEQ ID NOs: 1-249.

In one embodiment, the GCC agonist peptide is a peptide having the aminoacid sequence of any one of Formulas X-XVII (e.g. SEQ ID NO:87-98).

In some embodiments, GCC agonist peptides include peptides having theamino acid sequence of Formula I, wherein at least one amino acid ofFormula I is a D-amino acid or a methylated amino acid and/or the aminoacid at position 16 is a serine. Preferably, the amino acid at position16 of Formula I is a D-amino acid or a methylated amino acid. Forexample, the amino acid at position 16 of Formula I is a d-leucine or ad-serine. Optionally, one or more of the amino acids at positions 1-3 ofFormula I are D-amino acids or methylated amino acids or a combinationof D-amino acids or methylated amino acids. For example, Asn¹, Asp² orGlu³ (or a combination thereof) of Formula I is a D-amino acid or amethylated amino acid. Preferably, the amino acid at position Xaa⁶ ofFormula I is a leucine, serine or tyrosine.

In alternative embodiments, GCC agonist peptides include peptides havingthe amino acid sequence of Formula II, wherein at least one amino acidof Formula II is a D-amino acid or a methylated amino acid. Preferably,the amino acid denoted by Xaa_(n2) of Formula II is a D-amino acid or amethylated amino acid. In some embodiments, the amino acid denoted byXaa_(n2) of Formula II is a leucine, a d-leucine, a serine, or ad-serine. Preferably, the one or more amino acids denoted by Xaa_(n1) ofFormula II is a D-amino acid or a methylated amino acid. Preferably, theamino acid at position Xaa⁶ of Formula II is a leucine, a serine, or atyrosine.

In some embodiments, GCC agonist peptides include peptides having theamino acid sequence of Formula III, wherein at least one amino acid ofFormula III is a D-amino acid or a methylated amino acid and/or Maa isnot a cysteine. Preferably, the amino acid denoted by Xaa_(n2) ofFormula III is a D-amino acid or a methylated amino acid. In someembodiments the amino acid denoted by Xaa_(n2) of Formula III is aleucine, a d-leucine, a serine, or a d-serine. Preferably, the one ormore amino acids denoted by Xaa_(n1) of Formula III is a D-amino acid ora methylated amino acid. Preferably, the amino acid at position Xaa⁶ ofFormula III is a leucine, a serine, or a tyrosine.

In other embodiments, GCC agonist peptides include peptides having theamino acid sequence of Formula IV, wherein at least one amino acid ofFormula IV is a D-amino acid or a methylated amino acid, and/or Maa isnot a cysteine. Preferably, the Xaa_(n2) of Formula IV is a D-amino acidor a methylated amino acid. In some embodiments, the amino acid denotedby Xaa_(n2) of Formula IV is a leucine, a d-leucine, a serine, or ad-serine. Preferably, the one or more of the amino acids denoted byXaa_(n1) of Formula IV is a D-amino acid or a methylated amino acid.Preferably, the amino acid denoted Xaa⁶ of Formula IV is a leucine, aserine, or a tyrosine.

In further embodiments, GCC agonist peptides include peptides having theamino acid sequence of Formula V, wherein at at least one amino acid ofFormula V is a D-amino acid or a methylated amino acid. Preferably, theamino acid at position 16 of Formula V is a D-amino acid or a methylatedamino acid. For example, the amino acid at position 16 (i.e., Xaa¹⁶) ofFormula V is a d-leucine or a d-serine. Optionally, one or more of theamino acids at position 1-3 of Formula V are D-amino acids or methylatedamino acids or a combination of D-amino acids or methylated amino acids.For example, Asn¹, Asp² or Glu³ (or a combination thereof) of Formula Vis a D-amino acids or a methylated amino acid. Preferably, the aminoacid denoted at Xaa⁶ of Formula V is a leucine, a serine, or a tyrosine.

In additional embodiments, GCC agonist peptides include peptides havingthe amino acid sequence of Formula VI, VII, VIII, or IX. Preferably, theamino acid at position 6 of Formula VI, VII, VIII, or IX is a leucine, aserine, or a tyrosine. In some aspects the amino acid at position 16 ofFormula VI, VII, VIII, or IX is a leucine or a serine. Preferably, theamino acid at position 16 of Formula V is a D-amino acid or a methylatedamino acid.

In additional embodiments, GCC agonist peptides include peptides havingthe amino acid sequence of Formula X, XI, XII, XIII, XIV, XV, XVI orXVII. Optionally, one or more amino acids of Formulas X, XI, XII, XIII,XIV, XV, XVI or XVII is a D-amino acid or a methylated amino acid.Preferably, the amino acid at the carboxy terminus of the peptidesaccording to Formulas X, XI, XII, XIII, XIV, XV, XVI or XVII is aD-amino acid or a methylated amino acid. For example the the amino acidat the carboxy terminus of the peptides according to Formulas X, XI,XII, XIII, XIV, XV, XVI or XVII is a D-tyrosine.

Preferably, the amino acid denoted by Xaa⁶ of Formula XIV is a tyrosine,phenyalanine or a serine. Most preferably the amino acid denoted by Xaa⁶of Formula XIV is a phenyalanine or a serine. Preferably, the amino aciddenoted by Xaa⁴ of Formula XV, XVI or XVII is a tyrosine, aphenyalanine, or a serine. Most preferably, the amino acid position Xaa⁴of Formula V, XVI or XVII is a phenyalanine or a serine.

In some embodiments, GCRA peptides include peptides containing the aminoacid sequence of Formula XVIII. Preferably, the amino acid at position 1of Formula XVIII is a glutamic acid, aspartic acid, glutamine or lysine.Preferably, the amino acid at position 2 and 3 of Formula XVIII is aglutamic acid, or an aspartic acid. Preferably, the amino acid atposition 5 a glutamic acid. Preferably, the amino acid at position 6 ofFormula XVIII is an isoleucine, valine, serine, threonine or tyrosine.Preferably, the amino acid at position 8 of Formula XVIII is a valine orisoleucine. Preferably, the amino acid at position 9 of Formula XVIII isa an asparagine. Preferably, the amino acid at position 10 of FormulaXVIII is a valine or an methionine. Preferably, the amino acid atposition 11 of Formula XVIII is an alanine. Preferably, the amino acidat position 13 of Formula XVIII is a threonine. Preferably, the aminoacid at position 14 of Formula XVIII is a glycine. Preferably, the aminoacid at position 16 of Formula XVIII is a leucine, serine or threonine

In alternative embodiments, GCRA peptides include peptides containingthe amino acid sequence of Formula XIX. Preferably, the amino acid atposition 1 of Formula XIX is a serine or asparagine. Preferably, theamino acid at position 2 of Formula XIX is a histidine or an asparticacid. Preferably, the amino acid at position 3 of Formula XIX is athreonine or a glutamic acid. Preferably, the amino acid at position 5of Formula XIX is a glutamic acid. Preferably, the amino acid atposition 6 of Formula XIX is an isoleucine, leucine, valine or tyrosine.Preferably, the amino acid at position 8, 10, 11, or 13 of Formula XIXis a alanine. Preferably, the amino acid at position 9 of Formula XIX isan asparagine or a phenylalanine. Preferably, the amino acid at position14 of Formula XIX is a glycine.

In further embodiments, GCRA peptides include peptides containing theamino acid sequence of Formula XX. Preferably, the amino acid atposition 1 of Formula XX is a glutamine. Preferably, the amino acid atposition 2 or 3 of Formula XX is a glutamic acid or a aspartic acid.Preferably, the amino acid at position 5 of Formula XX is a glutamicacid.

Preferably, the amino acid at position 6 of Formula XX is threonine,glutamine, tyrosine, isoleucine, or leucine. Preferably, the amino acidat position 8 of Formula XX is isoleucine or valine. Preferably, theamino acid at position 9 of Formula XX is asparagine. Preferably, theamino acid at position 10 of Formula XX is methionine or valine.Preferably, the amino acid at position 11 of Formula XX is alanine.Preferably, the amino acid at position 13 of Formula XX is a threonione.Preferably, the amino acid at position 1 of Formula XX is a glycine.Preferably, the amino acid at position 15 of Formula XX is a tyrosine.Optionally, the amino acid at position 15 of Formula XX is two aminoacid in length and is Cysteine (Cys), Penicillamine (Pen) homocysteine,or 3-mercaptoproline and serine, leucine or threonine.

In certain embodiments, one or more amino acids of the GCC agonistpeptides are replaced by a non-naturally occurring amino acid or anaturally or non-naturally occurring amino acid analog. Such amino acidsand amino acid analogs are known in the art. See, for example, Hunt,“The Non-Protein Amino Acids,” in Chemistry and Biochemistry of theAmino Acids, Barrett, Chapman and Hall, 1985. In some embodiments, anamino acid is replaced by a naturally-occurring, non-essential aminoacid, e.g., taurine. Non-limiting examples of naturally occurring aminoacids that can be replaced by non-protein amino acids include thefollowing: (1) an aromatic amino acid can be replaced by3,4-dihydroxy-L-phenylalanine, 3-iodo-L-tyrosine, triiodothyronine,L-thyroxine, phenylglycine (Phg) or nor-tyrosine (norTyr); (2) Phg andnorTyr and other amino acids including Phe and Tyr can be substitutedby, e.g., a halogen, —CH3, —OH, —CH2NH3, —C(O)H, —CH2CH3, —CN,—CH2CH2CH3, —SH, or another group; (3) glutamine residues can besubstituted with gamma-Hydroxy-Glu or gamma-Carboxy-Glu; (4) tyrosineresidues can be substituted with an alpha substituted amino acid such asL-alpha-methylphenylalanine or by analogues such as: 3-Amino-Tyr;Tyr(CH3); Tyr(PO3(CH3)2); Tyr(SO3H); beta-Cyclohexyl-Ala;beta-(1-Cyclopentenyl)-Ala; beta-Cyclopentyl-Ala; beta-Cyclopropyl-Ala;beta-Quinolyl-Ala; beta-(2-Thiazolyl)-Ala; beta-(Triazole-1-yl)-Ala;beta-(2-Pyridyl)-Ala; beta-(3-Pyridyl)-Ala; Amino-Phe; Fluoro-Phe;Cyclohexyl-Gly; tBu-Gly; beta-(3-benzothienyl)-Ala;beta-(2-thienyl)-Ala; 5-Methyl-Trp; and A-Methyl-Trp; (5) prolineresidues can be substituted with homopro (L-pipecolic acid);hydroxy-Pro; 3,4-Dehydro-Pro; 4-fluoro-Pro; or alpha-methyl-Pro or anN(alpha)-C(alpha) cyclized amino acid analogues with the structure: n=0,1, 2, 3; and (6) alanine residues can be substituted withalpha-substitued or N-methylated amino acid such as alpha-aminoisobutyric acid (aib), L/D-alpha-ethylalanine (L/D-isovaline),L/D-methylvaline, or L/D-alpha-methylleucine or a non-natural amino acidsuch as beta-fluoro-Ala. Alanine can also be substituted with: n=0, 1,2, 3 Glycine residues can be substituted with alpha-amino isobutyricacid (aib) or L/D-alpha-ethylalanine (L/D-isovaline).

Further examples of non-natural amino acids include: an unnatural analogof tyrosine; an unnatural analogue of glutamine; an unnatural analogueof phenylalanine; an unnatural analogue of serine; an unnatural analogueof threonine; an alkyl, aryl, acyl, azido, cyano, halo, hydrazine,hydrazide, hydroxyl, alkenyl, alkynl, ether, thiol, sulfonyl, seleno,ester, thioacid, borate, boronate, phospho, phosphono, phosphine,heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or aminosubstituted amino acid, or any combination thereof; an amino acid with aphotoactivatable cross-linker; a spin-labeled amino acid; a fluorescentamino acid; an amino acid with a novel functional group; an amino acidthat covalently or noncovalently interacts with another molecule; ametal binding amino acid; an amino acid that is amidated at a site thatis not naturally amidated, a metal-containing amino acid; a radioactiveamino acid; a photocaged and/or photoisomerizable amino acid; a biotinor biotin-analogue containing amino acid; a glycosylated or carbohydratemodified amino acid; a keto containing amino acid; amino acidscomprising polyethylene glycol or polyether; a heavy atom substitutedamino acid (e.g., an amino acid containing deuterium, tritium, ¹³C, ¹⁵N,or ¹⁸O); a chemically cleavable or photocleavable amino acid; an aminoacid with an elongated side chain; an amino acid containing a toxicgroup; a sugar substituted amino acid, e.g., a sugar substituted serineor the like; a carbon-linked sugar-containing amino acid; a redox-activeamino acid; an α-hydroxy containing acid; an amino thio acid containingamino acid; an α, α disubstituted amino acid; a β-amino acid; a cyclicamino acid other than proline; an O-methyl-L-tyrosine; anL-3-(2-naphthyl)alanine; a 3-methyl-phenylalanine; aρ-acetyl-L-phenylalanine; an O-4-allyl-L-tyrosine; a4-propyl-L-tyrosine; a tri-O-acetyl-GlcNAc β-serine; an L-Dopa; afluorinated phenylalanine; an isopropyl-L-phenylalanine; ap-azido-L-phenylalanine; a p-acyl-L-phenylalanine; ap-benzoyl-L-phenylalanine; an L-phosphoserine; a phosphonoserine; aphosphonotyrosine; a p-iodo-phenylalanine; a 4-fluorophenylglycine; ap-bromophenylalanine; a p-amino-L-phenylalanine; anisopropyl-L-phenylalanine; L-3-(2-naphthyl)alanine;D-3-(2-naphthyl)alanine (dNal); an amino-, isopropyl-, orO-allyl-containing phenylalanine analogue; a dopa, 0-methyl-L-tyrosine;a glycosylated amino acid; a p-(propargyloxy)phenylalanine;dimethyl-Lysine; hydroxy-proline; mercaptopropionic acid; methyl-lysine;3-nitro-tyrosine; norleucine; pyro-glutamic acid; Z (Carbobenzoxyl);ε-Acetyl-Lysine; β-alanine; aminobenzoyl derivative; aminobutyric acid(Abu); citrulline; aminohexanoic acid; aminoisobutyric acid (AM);cyclohexylalanine; d-cyclohexylalanine; hydroxyproline; nitro-arginine;nitro-phenylalanine; nitro-tyrosine; norvaline; octahydroindolecarboxylate; ornithine (Orn); penicillamine (PEN);tetrahydroisoquinoline; acetamidomethyl protected amino acids andpegylated amino acids. Further examples of unnatural amino acids andamino acid analogs can be found in U.S. 20030108885, U.S. 20030082575,US20060019347 (paragraphs 410-418) and the references cited therein. Thepolypeptides of the invention can include further modificationsincluding those described in US20060019347, paragraph 589. Exempary GCCagonist peptides which include a non-naturally occurring amino acidinclude for example SP-368 and SP-369.

In some embodiments, the GCC agonist peptides are cyclic peptides. GCCagonist cyclic peptides can be prepared by methods known in the art. Forexample, macrocyclization is often accomplished by forming an amide bondbetween the peptide N- and C-termini, between a side chain and the N- orC-terminus [e.g., with K₃Fe(CN)₆ at pH 8.5] (Samson et al.,Endocrinology, 137: 5182-5185 (1996)), or between two amino acid sidechains, such as cysteine. See, e.g., DeGrado, Adv Protein Chem, 39:51-124 (1988). In various embodiments, the GCC agonist peptides are[4,12; 7,15] bicycles.

In certain embodiments, one or both Cys residues which normally form adisulfide bond in a GCC agonist peptide are replaced with homocysteine,penicillamine, 3-mercaptoproline (Kolodziej et al. 1996 Int. J. Pept.Protein Res. 48:274), β, β dimethylcysteine (Hunt et al. 1993 Int. J.Pept. Protein Res. 42:249), or diaminopropionic acid (Smith et al. 1978J. Med. Chem. 2 1:117) to form alternative internal cross-links at thepositions of the normal disulfide bonds.

In certain embodiments, one or more disulfide bonds in a GCC agonistpeptide are replaced by alternative covalent cross-links, e.g., an amidelinkage (—CH₂CH(O)NHCH₂— or —CH₂NHCH(O)CH₂—), an ester linkage, athioester linkage, a lactam bridge, a carbamoyl linkage, a urea linkage,a thiourea linkage, a phosphonate ester linkage, an alkyl linkage(—CH₂CH₂CH₂CH₂—), an alkenyl linkage (—CH₂CH═CHCH₂—), an ether linkage(—CH₂CH₂OCH₂— or —CH₂OCH₂CH₂—), a thioether linkage (—CH₂CH₂SCH₂— or—CH₂SCH₂CH₂—), an amine linkage (—CH₂CH₂NHCH₂— or —CH₂NHCH₂CH₂—) or athioamide linkage (—CH₂CH(S)HNHCH₂— or —CH₂NHCH(S)CH₂—). For example,Ledu et al. (Proc. Natl. Acad. Sci. 100:11263-78, 2003) describe methodsfor preparing lactam and amide cross-links. Exemplary GCC agonistpeptides which include a lactam bridge include, for example, SP-370.

In certain embodiments, the GCC agonist peptides have one or moreconventional polypeptide bonds replaced by an alternative bond. Suchreplacements can increase the stability of the polypeptide. For example,replacement of the polypeptide bond between a residue amino terminal toan aromatic residue (e.g. Tyr, Phe, Trp) with an alternative bond canreduce cleavage by carboxy peptidases and may increase half-life in thedigestive tract. Bonds that can replace polypeptide bonds include: aretro-inverso bond (C(O)—NH instead of NH—C(O); a reduced amide bond(NH—CH₂); a thiomethylene bond (S—CH₂ or CH₂—S); an oxomethylene bond(O—CH₂ or CH₂—O); an ethylene bond (CH₂—CH₂); a thioamide bond(C(S)—NH); a trans-olefine bond (CH═CH); a fluoro substitutedtrans-olefme bond (CF═CH); a ketomethylene bond (C(O)—CHR or CHR—C(O)wherein R is H or CH₃; and a fluoro-ketomethylene bond (C(O)—CFR orCFR—C(O) wherein R is H or F or CH₃.

In certain embodiments, the GCC agonist peptides are modified usingstandard modifications. Modifications may occur at the amino (N-),carboxy (C-) terminus, internally or a combination of any of thepreceeding. In one aspect described herein, there may be more than onetype of modification on the polypeptide. Modifications include but arenot limited to: acetylation, amidation, biotinylation, cinnamoylation,farnesylation, formylation, myristoylation, palmitoylation,phosphorylation (Ser, Tyr or Thr), stearoylation, succinylation,sulfurylation and cyclisation (via disulfide bridges or amidecyclisation), and modification by Cys3 or Cys5. The GCC agonist peptidesdescribed herein may also be modified by 2, 4-dinitrophenyl (DNP),DNP-lysine, modification by 7-Amino-4-methyl-coumarin (AMC),flourescein, NBD (7-Nitrobenz-2-Oxa-1,3-Diazole), p-nitro-anilide,rhodamine B, EDANS (5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid),dabcyl, dabsyl, dansyl, texas red, FMOC, and Tamra(Tetramethylrhodamine). The GCC agonist peptides described herein mayalso be conjugated to, for example, polyethylene glycol (PEG); alkylgroups (e.g., C1-C20 straight or branched alkyl groups); fatty acidradicals; combinations of PEG, alkyl groups and fatty acid radicals(See, U.S. Pat. No. 6,309,633; Soltero et al., 2001 Innovations inPharmaceutical Technology 106-110); BSA and KLH (Keyhole LimpetHemocyanin). The addition of PEG and other polymers which can be used tomodify polypeptides of the invention is described in US20060 19347section IX.

A GCC agonist peptide can also be a derivatives of a GCC agonist peptidedescribed herein. For example, a derivative includes hybrid and modifiedforms of GCC agonist peptides in which certain amino acids have beendeleted or replaced. A modification may also include glycosylation.Preferrably, where the modification is an amino acid substitution, it isa conservative substitution at one or more positions that are predictedto be non-essential amino acid residues for the biological activity ofthe peptide. A “conservative substitution” is one in which the aminoacid residue is replaced with an amino acid residue having a similarside chain. Families of amino acid residues having similar side chainshave been defined in the art. These families include amino acids withbasic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine).

In one embodiment, a GCC agonist peptide described herein is subjectedto random mutagenesis in order to identify mutants having biologicalactivity.

In one embodiment, the GCC agonist peptide is substantially homologousis a GCC agonist peptide described herein. Such substantially homologouspeptides can be isolated by virtue of cross-reactivity with antibodiesto a GCC agonist peptide described herein.

Further examples of GCC agonist peptides that can be used in the methodsand formulations of the invention are found in Tables I-VII below.

1.2.2 Preparation of GCC Agonist Peptides

GCC agonist peptides can be prepared using art recognized techniquessuch as molecular cloning, peptide synthesis, or site-directedmutagenesis.

Peptide synthesis can be performed using standard solution phase orsolid phase peptide synthesis techniques in which a peptide linkageoccurs through the direct condensation of the amino group of one aminoacid with the carboxy group of the other amino acid with the eliminationof a water molecule. Peptide bond synthesis by direct condensation, asformulated above, requires suppression of the reactive character of theamino group of the first and of the carboxyl group of the second aminoacid. The masking substituents must permit their ready removal, withoutinducing breakdown of the labile peptide molecule.

In solution phase synthesis, a wide variety of coupling methods andprotecting groups may be used (See, Gross and Meienhofer, eds., “ThePeptides: Analysis, Synthesis, Biology,” Vol. 1-4 (Academic Press,1979); Bodansky and Bodansky, “The Practice of Peptide Synthesis,” 2ded. (Springer Verlag, 1994)). In addition, intermediate purification andlinear scale up are possible. Those of ordinary skill in the art willappreciate that solution synthesis requires consideration of main chainand side chain protecting groups and activation method. In addition,careful segment selection is necessary to minimize racemization duringsegment condensation. Solubility considerations are also a factor. Solidphase peptide synthesis uses an insoluble polymer for support duringorganic synthesis. The polymer-supported peptide chain permits the useof simple washing and filtration steps instead of laboriouspurifications at intermediate steps. Solid-phase peptide synthesis maygenerally be performed according to the method of Merrifield et al., J.Am. Chem. Soc., 1963, 85:2149, which involves assembling a linearpeptide chain on a resin support using protected amino acids. Solidphase peptide synthesis typically utilizes either the Boc or Fmocstrategy, which are well known in the art.

Those of ordinary skill in the art will recognize that, in solid phasesynthesis, deprotection and coupling reactions must go to completion andthe side-chain blocking groups must be stable throughout the synthesis.In addition, solid phase synthesis is generally most suitable whenpeptides are to be made on a small scale.

Acetylation of the N-terminal can be accomplished by reacting the finalpeptide with acetic anhydride before cleavage from the resin.C-amidation is accomplished using an appropriate resin such asmethylbenzhydrylamine resin using the Boc technology.

Alternatively the GCC agonist peptides are produced by modern cloningtechniques For example, the GCC agonist peptides are produced either inbacteria including, without limitation, E. coli, or in other existingsystems for polypeptide or protein production (e.g., Bacillus subtilis,baculovirus expression systems using Drosophila Sf9 cells, yeast orfilamentous fungal expression systems, mammalian cell expressionsystems), or they can be chemically synthesized. If the GCC agonistpeptide or variant peptide is to be produced in bacteria, e.g., E. coli,the nucleic acid molecule encoding the polypeptide may also encode aleader sequence that permits the secretion of the mature polypeptidefrom the cell. Thus, the sequence encoding the polypeptide can includethe pre sequence and the pro sequence of, for example, anaturally-occurring bacterial ST polypeptide. The secreted, maturepolypeptide can be purified from the culture medium.

The sequence encoding a GCC agonist peptide described herein can beinserted into a vector capable of delivering and maintaining the nucleicacid molecule in a bacterial cell. The DNA molecule may be inserted intoan autonomously replicating vector (suitable vectors include, forexample, pGEM3Z and pcDNA3, and derivatives thereof). The vector nucleicacid may be a bacterial or bacteriophage DNA such as bacteriophagelambda or M13 and derivatives thereof. Construction of a vectorcontaining a nucleic acid described herein can be followed bytransformation of a host cell such as a bacterium. Suitable bacterialhosts include but are not limited to, E. coli, B subtilis, Pseudomonas,Salmonella. The genetic construct also includes, in addition to theencoding nucleic acid molecule, elements that allow expression, such asa promoter and regulatory sequences. The expression vectors may containtranscriptional control sequences that control transcriptionalinitiation, such as promoter, enhancer, operator, and repressorsequences.

A variety of transcriptional control sequences are well known to thosein the art. The expression vector can also include a translationregulatory sequence (e.g., an untranslated 5′ sequence, an untranslated3′ sequence, or an internal ribosome entry site). The vector can becapable of autonomous replication or it can integrate into host DNA toensure stability during polypeptide production.

The protein coding sequence that includes a GCC agonist peptidedescribed herein can also be fused to a nucleic acid encoding apolypeptide affinity tag, e.g., glutathione S-transferase (GST), maltoseE binding protein, protein A, FLAG tag, hexa-histidine, myc tag or theinfluenza HA tag, in order to facilitate purification. The affinity tagor reporter fusion joins the reading frame of the polypeptide ofinterest to the reading frame of the gene encoding the affinity tag suchthat a translational fusion is generated. Expression of the fusion generesults in translation of a single polypeptide that includes both thepolypeptide of interest and the affinity tag. In some instances whereaffinity tags are utilized, DNA sequence encoding a protease recognitionsite will be fused between the reading frames for the affinity tag andthe polypeptide of interest.

Genetic constructs and methods suitable for production of immature andmature forms of the GCC agonist peptides and variants described hereinin protein expression systems other than bacteria, and well known tothose skilled in the art, can also be used to produce polypeptides in abiological system.

The peptides disclosed herein may be modified by attachment of a secondmolecule that confers a desired property upon the peptide, such asincreased half-life in the body, for example, pegylation. Suchmodifications also fall within the scope of the term “variant” as usedherein.

TABLE I GCRA Peptides (SP-304 and Derivatives) SEQ Position of ID NameDisulfide bonds Structure NO SP-304 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶  1 SP-326 C3:C11, C6:C14Asp¹-Glu²-Cys³-Glu⁴-Leu⁵-Cys⁶-Val⁷-Asn⁸-Val⁹-Ala¹⁰-Cys¹¹-Thr¹²-Gly¹³-Cys¹⁴-Leu¹⁵  2 SP-327 C3:C11, C6:C14Asp¹-Glu²-Cys³-Glu⁴-Leu⁵-Cys⁶-Val⁷-Asn⁸-Val⁹-Ala¹⁰-Cys¹¹-Thr¹²-Gly¹³-Cys¹⁴  3 SP-328 C2:C10, C5:C13Glu¹-Cys²-Glu³-Leu⁴-Cys⁵-Val⁶-Asn⁷-Val⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-Leu¹⁴  4 SP-329 C2:C10, C5:C13Glu¹-Cys²-Glu³-Leu⁴-Cys⁵-Val⁶-Asn⁷-Val⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³   5SP-330 C1:C9, C4:C12Cys¹-Glu²-Leu³-Cys⁴-Val⁵-Asn⁶-Val⁷-Ala⁸-Cys⁹-Thr¹⁰-Gly¹¹-Cys¹²-Leu¹³   6SP-331 C1:C9, C4:C12Cys¹-Glu²-Leu³-Cys⁴-Val⁵-Asn⁶-Val⁷-Ala⁸-Cys⁹-Thr¹⁰-Gly¹¹-Cys¹²   7 SP332C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶  8 SP-333 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶  9 SP-334 C4:C12, C7:C15dAsn¹-dAsp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶ 10 SP-335 C4:C12, C7:C15dAsn¹-dAsp²-dGlu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶ 11 SP-336 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶ 12 SP-337 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-dLeu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶ 13 SP-338 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵ 14 SP-342 C4:C12, C7:C15PEG3-Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG3 15 SP-343 C4:C12, C7:C15PEG3-dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG3 16 SP-344 C4:C12, C7:C15PEG3-dAsn¹-dAsp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG3 17 SP-347 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG3 18 SP-348 C4:C12, C7:C15PEG3-Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶ 19 SP-350 C4:C12, C7:C15PEG3-dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶ 20 SP-352 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG3 21 SP-358 C4:C12, C7:C15PEG3-dAsn¹-dAsp²-dGlu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶- 22 PEG3 SP-359 C4:C12, C7:C15PEG3-dAsn¹-dAsp²-dGlu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶ 23 SP-360 C4:C12, C7:C15dAsn¹-dAsp²-dGlu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG3 24 SP-361 C4:C12, C7:C15dAsn¹-dAsp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG3 25 SP-362 C4:C12, C7:C15PEG3-dAsn¹-dAsp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶ 26 SP-368 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dNal¹⁶ 27 SP-369 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-AIB⁸-Asn⁹-AIB¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶ 28 SP-370 C4:C12, 7:15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Asp[Lactam]⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Orn¹⁵-dLeu¹ 29 SP-371 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶ 30 SP-372 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶ 31 N1 C4:C12, C7:C15PEG3-dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG3 32 N2 C4:C12, C7:C15PEG3-dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶ 33 N3 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG3 34 N4 C4:C12, C7:C15PEG3-dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG3 35 N5 C4:C12, C7:C15PEG3-dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶ 36 N6 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG3 37 N7 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶ 38 N8 C4:C12, C7:C15PEG3-Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶-PEG3 39 N9 C4:C12, C7:C15PEG3-Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶ 40 N10 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶-PEG3 41 N11 C4:C12, C7:C15PEG3-Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dSer¹⁶-PEG3 42 N12 C4:C12, C7:C15PEG3-Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dSer¹⁶ 43 N13 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dSer¹⁶-PEG3 44 Formula I C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Aaa¹¹-Cys¹²-Aaa¹³-Xaa¹⁴-Cys¹⁵-Xaa¹⁶ 45 Formula II C4:C12, C7:C15Xaa_(n1)-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-Xaa_(n2) 46 Formula 4:12, 7:15Xaa_(n1)-Maa⁴-Glu⁵-Xaa⁶-Maa⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Maa¹²-Thr¹³-Gly¹⁴-Maa¹⁵-Xaa_(n2) 47 III Formula 4:12, 7:15Xaa_(n1)-Maa⁴-Xaa⁵-Xaa⁶-Maa⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Maa¹²-Xaa¹³-Xaa¹⁴-Maa¹⁵-Xaa_(n2) 48 IV Formula V C4:C12, C7:C15Asn¹-Asp²-Asp³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Asn⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-Xaa¹⁶ 49 Formula C4:C12, C7:C15dAsn¹-Glu²-Glu³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Asn⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-d-Xaa¹⁶ 50 VI Formula C4:C12, C7:C15dAsn¹-dGlu²-Asp³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Asn⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-d-Xaa¹⁶ 51 VII Formula C4:C12, C7:C15dAsn¹-dAsp²-Glu³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Asn⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-d-Xaa¹⁶ 52 VII Formula C4:C12, C7:C15dAsn¹-dAsp²-dGlu³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Tyr⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-d-Xaa¹⁶ 53 VIII Formula C4:C12, C7:C15dAsn¹-dGlu²-dGlu³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Tyr⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-d-Xaa¹⁶ 54 IX

TABLE II Linaclotide and Derivatives Position of SEQ ID NameDisulfide Bonds Structure NO: SP-339 C1:C6, C2:C10, C5:C13Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-Tyr¹⁴55 (linaclotide) SP-340 C1:C6, C2:C10, C5:C13Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³ 56SP-349 C1:C6, C2:C10, C5:C13PEG3-Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-Tyr¹⁴-57 PEG3 SP-353 C3:C8, C4:C12, C7:C15Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-58 Tyr¹⁶ SP-354 C3:C8, C4:C12, C7:C15Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Phe⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-59 Tyr¹⁶ SP-355 C1:C6, C2:C10, C5:C13Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-dTyr¹⁴60 SP-357 C1:C6, C2:C10, C5:C13PEG3-Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-Tyr¹⁴61 SP-374 C3:C8, C4:C12, C7:C15Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-62 Tyr¹⁶ SP-375 C3:C8, C4:C12, C7:C15Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-63 dTyr¹⁶ SP-376 C3:C8, C4:C12, C7:C15dAsn¹-Phe²-Cys³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-64 Tyr¹⁶ SP-377 C3:C8, C4:C12, C7:C15dAsn¹-Phe²-Cys³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-65 dTyr¹⁶ SP-378 C3:C8, C4:C12, C7:C15Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-66 dTyr¹⁶ SP-379 C3:C8, C4:C12, C7:C15dAsn¹-Phe²-Cys³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-67 Tyr¹⁶ SP-380 C3:C8, C4:C12, C7:C15dAsn¹-Phe²-Cys³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-68 dTyr¹⁶ SP-381 C3:C8, C4:C12, C7:15Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Phe⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-69 dTyr¹⁶ SP-382 C3:C8, C4:C12, C7:15dAsn¹-Phe²-Cys³-Cys⁴-Glu⁵-Phe⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-70 Tyr¹⁶ SP-383 C3:C8, C4:C12, C7:15dAsn¹-Phe²-Cys³-Cys⁴-Glu⁵-Phe⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-71 dTyr¹⁶ SP384 C1:C6, C2:C10, C5:C13Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-Tyr¹⁴-PEG372 N14 C1:C6, C2:C10, C5:C13PEG3-Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-PEG373 N15 C1:C6, C2:C10, C5:C13PEG3-Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³74 N16 C1:C6, C2:C10, C5:C13Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-PEG375 N17 C3:C8, C4:C12, C7:C15PEG3-Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-76 Cys¹⁵-Tyr¹⁶-PEG3 N18 C3:C8, C4:C12, C7:C15PEG3-Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-77 Cys¹⁵-Tyr¹⁶ N19 C3:C8, C4:C12, C7:C15Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-78 Tyr¹⁶-PEG3 N20 C3:C8, C4:C12, C7:C15PEG3-Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Phe⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-79 Cys¹⁵-Tyr¹⁶-PEG3 N21 C3:C8, C4:C12, C7:C15PEG3-Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Phe⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-80 Cys¹⁵-Tyr¹⁶ N22 C3:C8, C4:C12, C7:C15Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Phe⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-81 Tyr¹⁶-PEG3 N23 C3:C8, C4:C12, C7:C15PEG3-Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-82 Cys¹⁵-Tyr¹⁶-PEG3 N24 C3:C8, C4:C12, C7:C15PEG3-Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-83 Cys¹⁵-Tyr¹⁶ N25 C3:C8, C4:C12, C7:C15Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-84 Tyr¹⁶-PEG3 N26 C1:C6, C2:C10, C5:C13Cys¹-Cys²-Glu³-Ser⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-Tyr¹⁴85 N27 C1:C6, C2:C10, C5:C13Cys¹-Cys²-Glu³-Phe⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-Tyr¹⁴86 N28 C1:C6, C2:C10, C5:C13Cys¹-Cys²-Glu³-Ser⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³- 87N29 C1:C6, C2:C10, C5:C13Cys¹-Cys²-Glu³-Phe⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³ 88N30 1:6, 2:10, 5:13Pen¹-Pen²-Glu³-Tyr⁴-Pen⁵-Pen⁶-Asn⁷-Pro⁸-Ala⁹-Pen¹⁰-Thr¹¹-Gly¹²-Pen¹³-Tyr¹⁴89 N31 1:6, 2:10, 5:13Pen¹-Pen²-Glu³-Tyr⁴-Pen⁵-Pen⁶-Asn⁷-Pro⁸-Ala⁹-Pen¹⁰-Thr¹¹-Gly¹²-Pen¹³ 90Formula X C9:C14, C10:C18,Xaa¹-Xaa²-Xaa³-Xaa⁴-Xaa⁵-Xaa⁶-Asn⁷-Tyr⁸-Cys⁹-Cys¹⁰-Xaa¹¹-Tyr¹²-Cys¹³-Cys¹⁴-Xaa¹⁵-91 C13:C21 Xaa¹⁶-Xaa¹⁷-Cys¹⁸-Xaa¹⁹-Xaa²⁰-Cys²¹-Xaa²² Formula XIC9:C14, C10:C18,Xaa¹-Xaa²-Xaa³-Xaa⁴-Xaa⁵-Xaa⁶-Asn⁷-Phe⁸-Cys⁹-Cys¹⁰-Xaa¹¹-Phe¹²-Cys¹³-Cys¹⁴-Xaa¹⁵-92 C13:C21 Xaa¹⁶-Xaa¹⁷-Cys¹⁸-Xaa¹⁹-Xaa²⁰-Cys²¹-Xaa²² Formula XIIC3:C8, C4:C12, C7:C15Asn¹-Phe²-Cys³-Cys⁴-Xaa⁵-Phe⁶-Cys⁷-Cys⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-93 Xaa¹⁶ Formula XIII 3:8, 4:12, 7:15Asn¹-Phe²-Pen³-Cys⁴-Xaa⁵-Phe⁶-Cys⁷-Pen⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-94 Xaa¹⁶ Formula XIV 3:8, 4:12, 7:15Asn¹-Phe²-Maa³-Maa⁴-Xaa⁵-Xaa⁶-Maa⁷-Maa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Maa¹²-Xaa¹³-Xaa¹⁴-95 Maa¹⁵-Xaa¹⁶ Formula XV 1:6, 2:10, 5:13Maa¹-Maa²-Glu³-Xaa⁴-Maa⁵-Maa⁶-Asn⁷-Pro⁸-Ala⁹-Maa¹⁰-Thr¹¹-Gly¹²-Maa¹³-Tyr¹⁴96 Formula XVI 1:6, 2:10, 5:13Maa¹-Maa²-Glu³-Xaa⁴-Maa⁵-Maa⁶-Asn⁷-Pro⁸-Ala⁹-Maa¹⁰-Thr¹¹-Gly¹²-Maa¹³ 97Formula XVII 1:6, 2:10, 5:13Xaa_(n3)-Maa¹-Maa²-Xaa³-Xaa⁴-Maa⁵-Maa⁶-Xaa⁷-Xaa⁸-Xaa⁹-Maa¹⁰-Xaa¹¹-Xaa¹²-Maa¹³-98 Xaa_(n2)

TABLE III GCRA Peptides Position of SEQ ID  Name Disulfide bondsStructure NO: SP-363 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³- 99 Gly¹⁴-Cys¹⁵-dLeu-AMIDE¹⁶ SP-364 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-100 Gly¹⁴-Cys¹⁵-dSer¹⁶ SP-365 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-101 Gly¹⁴-Cys¹⁵-dSer-AMIDE¹⁶ SP-366 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-102 Gly¹⁴-Cys¹⁵-dTyr¹⁶ SP-367 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-103 Gly¹⁴-Cys¹⁵-dTyr-AMIDE¹⁶ SP-373 C4:C12, C7:C15Pyglu¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-104 Gly¹⁴-Cys¹⁵-dLeu-AMIDE¹⁶ SP-304 C4:C12, C7:C15PEG3-Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-105 di PEG Gly¹⁴-Cys¹⁵-Leu¹⁶-PEG3 SP-304 N- C4:C12, C7:C15PEG3-Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-106 PEG Gly¹⁴-Cys¹⁵-Leu¹⁶ SP-304 C- C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-107 PEG Cys¹⁵-Leu¹⁶-PEG3

TABLE IV SP-304 Analogs, Uroguanylin, and Uroguanylin AnalogsPosition of SEQ ID Name Disulfide bonds Structure NO Formula C4:C12,Xaa¹-Xaa²-Xaa³-Maa⁴-Xaa⁵-Xaa⁶-Maa⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Maa¹²-Xaa¹³-108 XVIII C7:C15 Xaa¹⁴-Maa¹⁵-Xaa¹⁶ Uro- C4:C12,Asn¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-109 guanylin C7:C15 Gly¹⁴-Cys¹⁵-Leu¹⁶ N32 C4:C12, C7:C15Glu¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-110 Gly¹⁴-Cys¹⁵-Leu¹⁶ N33 C4:C12, C7:C15Glu¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-111 Gly¹⁴-Cys¹⁵-Leu¹⁶ N34 C4:C12, C7:C15Glu¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-112 Gly¹⁴-Cys¹⁵-Leu¹⁶ N35 C4:C12, C7:C15Glu¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-113 Gly¹⁴-Cys¹⁵-Leu¹⁶ N36 C4:C12, C7:C15Asp¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-114 Gly¹⁴-Cys¹⁵-Leu¹⁶ N37 C4:C12, C7:C15Asp¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-115 Gly¹⁴-Cys¹⁵-Leu¹⁶ N38 C4:C12, C7:C15Asp¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-116 Gly¹⁴-Cys¹⁵-Leu¹⁶ N39 C4:C12, C7:C15Asp¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-117 Gly¹⁴-Cys¹⁵-Leu¹⁶ N40 C4:C12, C7:C15Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-118 Gly¹⁴-Cys¹⁵-Leu¹⁶ N41 C4:C12, C7:C15Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-119 Gly¹⁴-Cys¹⁵-Leu¹⁶ N42 C4:C12, C7:C15Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-120 Gly¹⁴-Cys¹⁵-Leu¹⁶ N43 C4:C12, C7:C15Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-121 Gly¹⁴-Cys¹⁵-Leu¹⁶ N44 C4:C12, C7:C15Lys¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-122 Gly¹⁴-Cys¹⁵-Leu¹⁶ N45 C4:C12, C7:C15Lys¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-123 Gly¹⁴-Cys¹⁵-Leu¹⁶ N46 C4:C12, C7:C15Lys¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-124 Gly¹⁴-Cys¹⁵-Leu¹⁶ N47 C4:C12, C7:C15Lys¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-125 Gly¹⁴-Cys¹⁵-Leu¹⁶ N48 C4:C12, C7:C15Glu¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-126 Gly¹⁴-Cys¹⁵-Leu¹⁶ N49 C4:C12, C7:C15Glu¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-127 Gly¹⁴-Cys¹⁵-Leu¹⁶ N50 C4:C12, C7:C15Glu¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-128 Gly¹⁴-Cys¹⁵-Leu¹⁶ N51 C4:C12, C7:C15Glu¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-129 Gly¹⁴-Cys¹⁵-Leu¹⁶ N52 C4:C12, C7:C15Asp¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-⁶130 Gly¹⁴-Cys¹⁵-Leu¹ N53 C4:C12, C7:C15Asp¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-131 Gly¹⁴-Cys¹⁵-Leu¹⁶ N54 C4:C12, C7:C15Asp¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-132 Gly¹⁴-Cys¹⁵-Leu¹⁶ N55 C4:C12, C7:C15Asp¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-133 Gly¹⁴-Cys¹⁵-Leu¹⁶ N56 C4:C12, C7:C15Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-134 Gly¹⁴-Cys¹⁵-Leu¹⁶ N57 C4:C12, C7:C15Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-135 Gly¹⁴-Cys¹⁵-Leu¹⁶ N58 C4:C12, C7:C15Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-136 Gly¹⁴-Cys¹⁵-Leu¹⁶ N59 C4:C12, C7:C15Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-137 Gly¹⁴-Cys¹⁵-Leu¹⁶ N60 C4:C12, C7:C15Lys¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-138 Gly¹⁴-Cys¹⁵-Leu¹⁶ N61 C4:C12, C7:C15Lys¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-139 Gly¹⁴-Cys¹⁵-Leu¹⁶ N62 C4:C12, C7:C15Lys¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-140 Gly¹⁴-Cys¹⁵-Leu¹⁶ N63 C4:C12, C7:C15Lys¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-141 Gly¹⁴-Cys¹⁵-Leu¹⁶ N65 C4:C12, C7:C15Glu¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-142 Gly¹⁴-Cys¹⁵-Leu¹⁶ N66 C4:C12, C7:C15Glu¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-143 Gly¹⁴-Cys¹⁵-Leu¹⁶ N67 C4:C12, C7:C15Glu¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-144 Gly¹⁴-Cys¹⁵-Leu¹⁶ N68 C4:C12, C7:C15Glu¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-145 Gly¹⁴-Cys¹⁵-Leu¹⁶ N69 C4:C12, C7:C15Asp¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-146 Gly¹⁴-Cys¹⁵-Leu¹⁶ N70 C4:C12, C7:C15Asp¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-147 Gly¹⁴-Cys¹⁵-Leu¹⁶ N71 C4:C12, C7:C15Asp¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-148 Gly¹⁴-Cys¹⁵-Leu¹⁶ N72 C4:C12, C7:C15Asp¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-149 Gly¹⁴-Cys¹⁵-Leu¹⁶ N73 C4:C12, C7:C15Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-150 Gly¹⁴-Cys¹⁵-Leu¹⁶ N74 C4:C12, C7:C15Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-151 Gly¹⁴-Cys¹⁵-Leu¹⁶ N75 C4:C12, C7:C15Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-152 Gly¹⁴-Cys¹⁵-Leu¹⁶ N76 C4:C12, C7:C15Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-153 Gly¹⁴-Cys¹⁵-Leu¹⁶ N77 C4:C12, C7:C15Lys¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-154 Gly¹⁴-Cys¹⁵-Leu¹⁶ N78 C4:C12, C7:C15Lys¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-155 Gly¹⁴-Cys¹⁵-Leu¹⁶ N79 C4:C12, C7:C15Lys¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-156 Gly¹⁴-Cys¹⁵-Leu¹⁶ N80 C4:C12, C7:C15Lys¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-157 Gly¹⁴-Cys¹⁵-Leu¹⁶ N81 C4:C12, C7:C15Glu¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-158 Gly¹⁴-Cys¹⁵-Leu¹⁶ N82 C4:C12, C7:C15Glu¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-159 Gly¹⁴-Cys¹⁵-Leu¹⁶ N83 C4:C12, C7:C15Glu¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-160 Gly¹⁴-Cys¹⁵-Leu¹⁶ N84 C4:C12, C7:C15Glu¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-161 Gly¹⁴-Cys¹⁵-Leu¹⁶ N85 C4:C12, C7:C15Asp¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-162 Gly¹⁴-Cys¹⁵-Leu¹⁶ N86 C4:C12, C7:C15Asp¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-163 Gly¹⁴-Cys¹⁵-Leu¹⁶ N87 C4:C12, C7:C15Asp¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-164 Gly¹⁴-Cys¹⁵-Leu¹⁶ N88 C4:C12, C7:C15Asp¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-165 Gly¹⁴-Cys¹⁵-Leu¹⁶ N89 C4:C12, C7:C15Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-166 Gly¹⁴-Cys¹⁵-Leu¹⁶ N90 C4:C12, C7:C15Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-167 Gly¹⁴-Cys¹⁵-Leu¹⁶ N91 C4:C12, C7:C15Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-168 Gly¹⁴-Cys¹⁵-Leu¹⁶ N92 C4:C12, C7:C15Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-169 Gly¹⁴-Cys¹⁵-Leu¹⁶ N93 C4:C12, C7:C15Lys¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-170 Gly¹⁴-Cys¹⁵-Leu¹⁶ N94 C4:C12, C7:C15Lys¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-171 Gly¹⁴-Cys¹⁵-Leu¹⁶ N95 C4:C12, C7:C15Lys¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-172 Gly¹⁴-Cys¹⁵-Leu¹⁶ N96 C4:C12, C7:C15Lys¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-173 Gly¹⁴-Cys¹⁵-Leu¹⁶

TABLE V Guanylin and Analogs Position of SEQ Disulfide ID Name bondsStructure NO Formula 4:12, 7:15Xaa¹-Xaa²-Xaa³-Maa⁴-Xaa⁵-Xaaa⁶-Maa⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Maa¹²-Xaa¹³-Xaa¹⁴-174 XIX Maa¹⁵ Guanylin C4:C12, C7:C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Phe⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-175 Cys¹⁵ N97 C4:C12, C7:C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵176 N98 C4:C12, C7:C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵177 N99 C4:C12, C7:C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Val⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵178 N100 C4:C12, C7:C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵179 N101 C4:C12, C7:C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵180 N102 C4:C12, C7:C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵181 N103 C4:C12, C7:C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Val⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵182 N104 C4:C12, C7:C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵183 N105 C4:C12, C7:C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵184 N106 C4:C12, C7:C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵185 N107 C4:C12, C7:C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Val⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵186 N108 C4:C12, C7:C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵187 N109 C4:C12, C7:C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵188 N110 C4:C12, C7:C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵189 N111 C4:C12, C7:C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Val⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵190 N112 C4:C12, C7:C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵191 N113 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵192 N114 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵193 N115 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Val⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵194 N116 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵195 N117 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵196 N118 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵197 N119 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Val⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵198 N120 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵199 N121 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵200 N122 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵201 N123 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Val⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵202 N124 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵203 N125 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵204 N126 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵205 N127 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Val⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵206 N128 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵207

TABLE VI Lymphoguanylin and Analogs Position of SEQ Disulfide ID Namebonds Structure NO Formula 4:12, 7:15Xaa¹-Xaa²-Xaa³-Maa⁴-Xaa⁵-Xaa⁶-Maa⁷-Xaa⁸-Xaa⁹-xaa¹⁰-Xaa¹¹-Maa¹²-Xaa¹³-Xaa¹⁴-Xaa_(n1)¹⁵ 208 XX Lympho- C4:C12Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵209 guanylin N129 C4:C12Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵210 N130 C4:C12Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵211 N131 C4:C12Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵212 N132 C4:C12Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵213 N133 C4:C12Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Glu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵214 N134 C4:C12Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Glu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵215 N135 C4:C12Glni-Asp²-Asp³-Cys⁴-Glu⁵-Glu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵216 N136 C4:C12Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Glu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵217 N137 C4:C12Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵218 N138 C4:C12Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵219 N139 C4:C12Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵220 N140 C4:C12Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵221 N141 C4:C12Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵222 N142 C4:C12Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵223 N143 C4:C12Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵224 N144 C4:C12Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵225 N145 C4:C12,Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-226 C7:C15 Ser¹⁶ N146 C4:C12,Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-227 C7:C15 Ser¹⁶ N147 C4:C12,Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-228 C7:C15 Ser¹⁶ N148 C4:C12,Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-229 C7:C15 Ser¹⁶ N149 C4:C12,Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Glu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-230 C7:C15 Ser¹⁶ N150 C4:C12,Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Glu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-231 C7:C15 Ser¹⁶ N151 C4:C12,Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Glu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-232 C7:C15 Ser¹⁶ N152 C4:C12,Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Glu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-233 C7:C15 Ser¹⁶ N153 C4:C12,Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-234 C7:C15 Ser¹⁶ N154 C4:C12,Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-235 C7:C15 Ser¹⁶ N155 C4:C12,Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-236 C7:C15 Ser¹⁶ N156 C4:C12,Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-237 C7:C15 Ser¹⁶ N157 C4:C12,Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-238 C7:C15 Ser¹⁶ N158 C4:C12,Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-239 C7:C15 Ser¹⁶ N159 C4:C12,Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-240 C7:C15 Ser¹⁶ N160 C4:C12,Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-241 C7:C15 Ser¹⁶

TABLE VII ST Peptide and Analogues Position of SEQ ID NameDisulfide bonds Structure NO ST C3:C8, C4:C12,Asn¹-Ser²-Ser³-Asn⁴-Ser⁵-Ser⁶-Asn⁷-Tyr⁸-Cys⁹-Cys¹⁰-Glu¹¹-Lys¹²-Cys¹³-242 Peptide C7:15 Cys¹⁴-Asn¹⁵-Pro¹⁶-Ala¹⁷-Cys¹⁸-Thr¹⁹-Gly²⁰-Cys²¹-Tyr²²N161 C3:C8, C4:C12,PEG3-Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²- 243C7:C15 Thr¹³-Gly¹⁴-Cys¹⁵-Tyr¹⁶-PEG3 N162 C3:C8, C4:C12,PEG3-Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²- 244C7:C15 Thr¹³-Gly¹⁴-Cys¹⁵-Tyr¹⁶ N163 C3:C8, C4:C12,Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-245 C7:C15 Gly¹⁴-Cys¹⁵-Tyr¹⁶-PEG3 N164 C3:C8, C4:C12,Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-246 C7:C15 Gly¹⁴-Cys¹⁵-Tyr¹⁶ N165 C3:C8, C4:C12,dAsn¹-Phe²-Cys³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-247 C7:C15 Gly¹⁴-Cys¹⁵-dTyr¹⁶ N166 C3:C8, C4:C12,Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-248 C7:C15 Gly¹⁴-Cys¹⁵-dTyr¹⁶ N167 C3:C8, C4:C12,dAsn¹-Phe²-Cys³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-249 C7:C15 Gly¹⁴-Cys¹⁵-Tyr¹⁶

1.3 Methods of Use

The invention provides methods for treating or preventinggastrointestinal disorders and increasing gastrointestinal motility in asubject in need thereof by administering an effective amount of a GCCagonist formulation to the subject. Non-limiting examples ofgastrointestinal disorders that can be treated or prevented according tothe methods of the invention include irritable bowel syndrome (IBS),non-ulcer dyspepsia, chronic intestinal pseudo-obstruction, functionaldyspepsia, colonic pseudo-obstruction, duodenogastric reflux,gastroesophageal reflux disease (GERD), ileus (e.g., post-operativeileus), gastroparesis, heartburn (high acidity in the GI tract),constipation (e.g., constipation associated with use of medications suchas opioids, osteoarthritis drugs, or osteoporosis drugs); post surigicalconstipation, constipation associated with neuropathic disorders,Crohn's disease, and ulcerative colitis.

The invention also provides methods for treating gastrointestinal cancerin a subject in need thereof by administering an effective amount of aGCC agonist formulation to the subject. Non-limiting examples ofgastrointestinal cancers that can be treated according to the methods ofthe invention include gastric cancer, esophageal cancer, pancreaticcancer, colorectal cancer, intestinal cancer, anal cancer, liver cancer,gallbladder cancer, or colon cancer.

Disorders are treated, prevented or alleviated by administering to asubject, e.g., a mammal such as a human in need thereof, atherapeutically effective dose of a GCC agonist peptide. The GCC agonistpeptides may be in a pharmaceutical composition in unit dose form,together with one or more pharmaceutically acceptable excipients. Theterm “unit dose form” refers to a single drug delivery entity, e.g., atablet, capsule, solution or inhalation formulation. The amount ofpeptide present should be sufficient to have a positive therapeuticeffect when administered to a patient (typically, between 10 μg and 3g). What constitutes a “positive therapeutic effect” will depend uponthe particular condition being treated and will include any significantimprovement in a condition readily recognized by one of skill in theart.

The GCC agonist formulations of the invention are particularly useful inthe treatment or prevention of diseases and disorders that benefit fromselective targeting of the GCC agonist to a region of thegastrointestinal tract. In one embodiment, the GCC agonist formulationtargets delivery of the GCC agonist to the duodenum or jejunum. Inaccordance with this embodiment, the GCC agonist formulation isparticularly useful for the treatment or prevention of one or more ofthe following: irritable bowel syndrome (preferably constipationpredominant), non-ulcer dyspepsia, chronic intestinalpseudo-obstruction, functional dyspepsia, colonic pseudo-obstruction,duodenogastric reflux, gastro esophageal reflux disease, chronicidiopathic constipation, gastroparesis, heartburn, gastric cancer, andH. pylori infection. In one embodiment, the GCC agonist formulation fortargeted delivery to the duodenum or jejunum comprises a pH dependentpolymer with a threshold pH between 4.5 and 6. In another embodiment,the GCC agonist formulation targets delivery of the GCC agonist to theileum or colon. In accordance with this embodiment, the GCC agonistformulation is particularly useful for the treatment or prevention ofone or more of the following: ileitis (post-operative ileitis), Crohn'sdisease, ulcerative colitis, terminal ileitis, and colon cancer. In oneembodiment, the GCC agonist formulation for targeted delivery to theileum or colon comprises a pH dependent polymer with a threshold pHbetween 6.5 and 7.5.

The specific dose of the GCC agonist to be administered in theformulations of the invention will depend on the nature of the diseaseor disorder to be treated or prevented as well as its severity. Otherfactors routinely used in determining the dosage for a particularsubject include the subject's body weight, general health, diet, andnatural history of disease. The route of administration and schedulingof administration will also be considered. In certain embodiments, aneffective dosage of a GCC agonist will typically be between about 10 μgand about 3 mg per kilogram body weight, preferably between about 10 μgand about 1 mg of the compound per kilogram body weight. In otherembodiments, the dosage of a GCC agonist will be effective to induceanti-inflammatory activity in the target tissue, especially the largeintestines (e.g., the terminal ileum and colon). In accordance with thisembodiment, the effective dosage of the GCC agonist will be from 0.01 mgto 10 mg per kilogram body weight. In a preferred embodiment, theeffective dosage is 0.01 mg/kg, 0.1 mg/kg, 1 mg/kg, 5 mg/kg, or 10 mg/kgbody weight. Adjustments in dosage will be made using methods that areroutine in the art and will be based upon the particular compositionbeing used and clinical considerations.

The GCC agonists for use in the methods described above are preferablyadministered orally. Dosage forms include solutions, suspensions,emulsions, tablets, and capsules.

The total daily dose can be administered to the patient in a singledose, or in multiple subdoses. Typically, subdoses can be administeredtwo to six times per day, preferably two to four times per day, and evenmore preferably two to three times per day.

The GCC agonists may be administered as either the sole active agent orin combination with one or more additional active agents. In all cases,additional active agents should be administered at a dosage that istherapeutically effective using the existing art as a guide. The GCCagonists may be administered in a single composition or sequentiallywith the one or more additional active agents. In one embodiment, theGCC agonist is administered in combination with one or more inhibitorsof cGMP dependent phosphodiesterase such as suldinac sulfone, zaprinast,motapizone, vardenafil, or sildenifil. In another embodiment, the GCCagonist is administered in combination with one or more chemotherapeuticagents. In another embodiment, the GCC agonist is administered incombination with one or more or anti-inflammatory drugs such as steroidsor non-steroidal anti-inflammatory drugs (NSAIDS), such as aspirin.

Combination therapy can be achieved by administering two or more agents,e.g., a GCC agonist peptide described herein and another compound, eachof which is formulated and administered separately, or by administeringtwo or more agents in a single formulation. Other combinations are alsoencompassed by combination therapy. For example, two agents can beformulated together and administered in conjunction with a separateformulation containing a third agent. While the two or more agents inthe combination therapy can be administered simultaneously, they neednot be. For example, administration of a first agent (or combination ofagents) can precede administration of a second agent (or combination ofagents) by minutes, hours, days, or weeks. Thus, the two or more agentscan be administered within minutes of each other or within 1, 2, 3, 6,9, 12, 15, 18, or 24 hours of each other or within 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 12, 14 days of each other or within 2, 3, 4, 5, 6, 7, 8, 9, or10 weeks of each other. In some cases even longer intervals arepossible. While in many cases it is desirable that the two or moreagents used in a combination therapy be present in within the patient'sbody at the same time, this need not be so.

The GCC agonist peptides described herein may be combined withphosphodiesterase inhibitors, e.g., sulindae sulfone, Zaprinast,sildenafil, vardenafil or tadalafil to further enhance levels of cGMP inthe target tissues or organs.

Combination therapy can also include two or more administrations of oneor more of the agents used in the combination. For example, if agent Xand agent Y are used in a combination, one could administer themsequentially in any combination one or more times, e.g., in the orderX-Y-X, X-X-Y, Y-X-Y, Y-Y-X, X-X-Y-Y, etc.

1.3.1 Exemplary Agents for Combination Therapy

The GCC agonist formulations of the invention may be administered aloneor in combination with one or more additional therapeutic agents as partof a therapeutic regimen for the treatment or prevention of agastrointestinal disease or disorder. In some embodiments, the GCCagonist formulation comprises one or more additional therapeutic agents.In other embodiments, the GCC agonist is formulated separately from theone or more additional therapeutic agents. In accordance with thisembodiment, the GCC agonist is administered either simultaneously,sequentially, or at a different time than the one or more additionaltherapeutic agents. In one embodiment, the GCC agonist formulation isadministered in combination with one or more additional therapeuticagents selected from the group consisting of phosphodiesteraseinhibitors, cyclic nucleotides (such as cGMP and cAMP), a laxative (suchas SENNA or METAMUCIL), a stool softner, an anti-tumor necrosis factoralpha therapy for IBD (such as REMICADE, ENBREL, or HUMIRA), andanti-inflammatory drugs (such as COX-2 inhibitors, sulfasalazine, 5-ASAderivatives and NSAIDS). In certain embodiments, the GCC agonistformulation is administered in combination with an effective dose of aninhibitor of cGMP-specific phosphodiesterase (cGMP-PDE) eitherconcurrently or sequentially with said GCC agonist. cGMP-PDE inhibitorsinclude, for example, suldinac sulfone, zaprinast, motapizone,vardenifil, and sildenafil. In another embodiment, the GCC agonistformulation is administered in combination with inhibitors of cyclicnucleotide transporters. Further examples of therapeutic agents that maybe administered in combination with the GCC agonist formulations of theinvention are given in the following sections.

1.3.1.1 Agents to Treat Gastrointestinal Cancers

The GCC agonist formulations described herein can be used in combinationwith one or more antitumor agents including but not limited toalkylating agents, epipodophyllotoxins, nitrosoureas, antimetabolites,vinca alkaloids, anthracycline antibiotics, nitrogen mustard agents, andthe like. Particular antitumor agents include tamoxifen, taxol,etoposide, and 5-fluorouracil. In one embodiment, the GCC agonistformulations are used in combination with an antiviral agent or amonoclonal antibody.

Non-limiting examples of antitumor agents that can be used incombination with the GCC agonist formulations of the invention for thetreatment of colon cancer include anti-proliferative agents, agents forDNA modification or repair, DNA synthesis inhibitors, DNA/RNAtranscription regulators, RNA processing inhibitors, agents that affectprotein expression, synthesis and stability, agents that affect proteinlocalization or their ability to exert their physiological action,agents that interfere with protein-protein or protein-nucleic acidinteractions, agents that act by RNA interference, receptor bindingmolecules of any chemical nature (including small molecules andantibodies), targeted toxins, enzyme activators, enzyme inhibitors, generegulators, HSP-90 inhibitors, molecules interfering with microtubulesor other cytoskeletal components or cell adhesion and motility, agentsfor phototherapy, and therapy adjuncts.

Representative anti-proliferative agents include N-acetyl-D-sphingosine(C.sub.2 ceramide), apigenin, berberine chloride,dichloromethylenediphosphonic acid disodium salt, 1oe-emodine, emodin,HA 14-1, N-hexanoyl-D-sphingosine (C.sub.6 ceramide),7b-hydroxycholesterol, 25-hydroxycholesterol, hyperforin, parthenolide,and rapamycin. Representative agents for DNA modification and repairinclude aphidicolin, bleomycin sulfate, carboplatin, carmustine,chlorambucil, cyclophosphamide monohydrate, cyclophosphamide monohydrateISOPAC®, cis-diammineplatinum(II) dichloride (Cisplatin), esculetin,melphalan, methoxyamine hydrochloride, mitomycin C, mitoxantronedihydrochloride, oxaliplatin, and streptozocin.

Representative DNA synthesis inhibitors include (.+−.)amethopterin(methotrexate), 3-amino-1,2,4-benzotriazine 1,4-dioxide, aminopterin,cytosine b-D-arabinofurdnoside (Ara-C), cytosine b-D-arabinofuranoside(Ara-C) hydrochloride, 2-fluoroadenine-9-b-D-arabinofuranoside(Fludarabine des-phosphate; F-ara-A), 5-fluoro-5′-deoxyuridine,5-fluorouracil, ganciclovir, hydroxyurea, 6-mercaptopurine, and6-thioguanine.

Representative DNA/RNA transcription regulators include actinomycin D,daunorubicin hydrochloride, 5,6-dichlorobenzimidazole1-b-D-ribofuranoside, doxorubicin hydrochloride, homoharringtonine, andidarubicin hydrochloride.

Representative enzyme activators and inhibitors include forskolin,DL-aminoglutethimide, apicidin, Bowman-Birk Inhibitor, butein,(S)-(+)-camptothecin, curcumin, (−)-deguelin, (−)-depudecin, doxycyclinehyclate, etoposide, formestane, fostriecin sodium salt, hispidin,2-imino-1-imidazolidineacetic acid (Cyclocreatine), oxamflatin,4-phenylbutyric acid, roscovitine, sodium valproate, trichostatin A,tyrphostin AG 34, tyrphostin AG 879, urinary trypsin inhibitor fragment,valproic acid (2-propylpentanoic acid), and XK469.

Representative gene regulators include 5-aza-2′-deoxycytidine,5-azacytidine, cholecalciferol (Vitamin D3), ciglitizone, cyproteroneacetate, 15-deoxy-D.sup.12,14-prostaglandin J.sub.2, epitestosterone,flutamide, glycyrrhizic acid ammonium salt (glycyrrhizin),4-hydroxytamoxifen, mifepristone, procainamide hydrochloride, raloxifenehydrochloride, all trans-retinal (vitamin A aldehyde), retinoic acid(vitamin A acid), 9-cis-retinoic acid, 13-cis-retinoic acid, retinoicacid p-hydroxyanilide, retinol (Vitamin A), tamoxifen, tamoxifen citratesalt, tetradecylthioacetic acid, and troglitazone.

Representative HSP-90 inhibitors include17-(allylamino)-17-demethoxygeldanamycin and geldanamycin.

Representative microtubule inhibitors include colchicines, dolastatin15, nocodazole, taxanes and in particular paclitaxel, podophyllotoxin,rhizoxin, vinblastine sulfate salt, vincristine sulfate salt, andvindesine sulfate salt and vinorelbine (Navelbine) ditartrate salt.

Representative agents for performing phototherapy include photoactiveporphyrin rings, hypericin, 5-methoxypsoralen, 8-methoxypsoralen,psoralen and ursodeoxycholic acid.

Representative agents used as therapy adjuncts include amifostine,4-amino-1,8-naphthalimide, brefeldin A, cimetidine, phosphomycindisodium salt, leuprolide (leuprorelin) acetate salt, luteinizinghormone-releasing hormone (LH-RH) acetate salt, lectin, papaverinehydrochloride, pifithrin-a, (−)-scopolamine hydrobromide, andthapsigargin.

The agents can also be anti-VEGF (vascular endothelial growth factor)agents, as such are known in the art. Several antibodies and smallmolecules are currently in clinical trials or have been approved thatfunction by inhibiting VEGF, such as Avastin (Bevacizumab), SU5416,SU11248 and BAY 43-9006. The agents can also be directed against growthfactor receptors such as those of the EGF/Erb-B family such as EGFReceptor (Iressa or Gefitinib, and Tarceva or Erlotinib), Erb-B2,receptor (Herceptin or Trastuzumab), other receptors (such as Rituximabor Rituxan/MabThera), tyrosine kinases, non-receptor tyrosine kinases,cellular serine/threonine kinases (including MAP kinases), and variousother proteins whose deregulation contribute to oncogenesis (such assmall/Ras family and large/heterotrimeric G proteins). Severalantibodies and small molecules targeting those molecules are currentlyat various stages of development (including approved for treatment or inclinical trials).

In a preferred embodiment, the invention provides a method for treatingcolon cancer in a subject in need thereof by administering to thesubject a GCC agonist formulation in combination with one or moreantitumor agent selected from the group consisting of paclitaxel,docetaxel, tamoxifen, vinorelbine, gemcitabine, cisplatin, etoposide,topotecan, irinotecan, anastrozole, rituximab, trastuzumab, fludarabine,cyclophosphamide, gentuzumab, carboplatin, interferons, and doxorubicin.In a particular embodiment the antitumor agent is paclitaxel. In afurther embodiment, the method further comprises an antitumor agentselected from the group consisting of 5-FU, doxorubicin, vinorelbine,cytoxan, and cisplatin.

1.3.1.2 Agents that Treat Crohn's Disease

In one embodiment, a GCC agonist formulation of the invention isadministered as part of a combination therapy with one or moreadditional therapeutic agents for the treatment of Crohn's disease.Non-limiting examples of the one or more additional therapeutic agentsinclude sulfasalazine and other mesalamine-containing drugs, generallyknown as 5-ASA agents, such as Asacol, Dipentum, or Pentasa, orinfliximab (REMICADE). In certain embodiments, the one or moreadditional agents is a corticosteroid or an immunosuppressive agent suchas 6-mercaptopurine or azathioprine. In another embodiment, the one ormore additional agents is an antidiarrheal agent such as diphenoxylate,loperamide, or codeine.

1.3.1.3 Agents that Treat Ulcerative Colitis

In one embodiment, a GCC agonist formulation of the invention isadministered as part of a combination therapy with one or moreadditional therapeutic agents for the treatment of ulcerative colitis.The agents that are used to treat ulcerative colitis overlap with thoseused to treat Chrohn's Disease. Non-limiting examples of the one or moreadditional therapeutic agents that can be used in combination with a GCCagonist formulation of the invention include aminosalicylates (drugsthat contain 5-aminosalicyclic acid (5-ASA)) such as sulfasalazine,olsalazine, mesalamine, and balsalazide. Other therapeutic agents thatcan be used include corticosteroids, such as prednisone andhydrocortisone, immunomodulators, such as azathioprine,6-mercapto-purine (6-MP), cytokines, interleukins, and lymphokines, andanti-TNF-alpha agents, including the thiazolidinediones or glitazonessuch as rosiglitazone and pioglitazone. In one emobidment, the one ormore additional therapeutic agents includes both cyclosporine A and 6-MPor azathioprine for the treatment of active, severe ulcerative colitis.

1.3.1.4 Agents that Treat Constipation/Irritable Bowel Syndrome

In one embodiment, a GCC agonist formulation of the invention isadministered as part of a combination therapy with one or moreadditional therapeutic agents for the treatment of constipation, such asthat associated with irritable bowel syndrome. Non-limiting examples ofthe one or more additional therapeutic agents include laxatives such asSENNA, MIRALAX, LACTULOSE, PEG, or calcium polycarbophil), stoolsofteners (such as mineral oil or COLACE), bulking agents (such asMETAMUCIL or bran), agents such as ZELNORM (also called tegaserod), andanticholinergic medications such as BENTYL and LEVSIN.

1.3.1.5 Agents for the Treatment of Postoperative Ileus

In one embodiment, a GCC agonist formulation of the invention isadministered as part of a combination therapy with one or moreadditional therapeutic agents for the treatment of postoperative ileus.Non-limiting examples of the one or more additional therapeutic agentsinclude ENTEREG (alvimopan; formerly called ado lor/ADL 8-2698),conivaptan, and related agents describes in U.S. Pat. No. 6,645,959.

1.3.1.6 Anti-Obesity Agents

In one embodiment, a GCC agonist formulation of the invention isadministered as part of a combination therapy with one or moreadditional therapeutic agents for the treatment of obesity. Non-limitingexamples of the one or more additional therapeutic agents include 1 1βHSD-I (11-beta hydroxy steroid dehydrogenase type 1) inhibitors, such asBVT 3498, BVT 2733,3-(1-adamantyl)-4-ethyl-5-(ethylthio)-4H-1,2,4-triazole,3-(1-adamantyl)-5-(3,4,5-trimethoxyphenyl)-4-methyl-4H-1,2,4-triazole,3-adamantanyl-4,5,6,7,8,9,10,11,12,3a-decahydro-1,2,4-triazolo[4,3-a][11]annulene, and those compounds disclosed in WO01/90091, WOO 1/90090,WOO 1/90092 and WO02/072084; 5HT antagonists such as those inWO03/037871, WO03/037887, and the like; 5HTIa modulators such ascarbidopa, benserazide and those disclosed in U.S. Pat. No. 6,207,699,WO03/031439, and the like; 5HT2c (serotonin receptor 2c) agonists, suchas BVT933, DPCA37215, IK264, PNU 22394, WAY161503, R-1065, SB 243213(Glaxo Smith Kline) and YM 348 and those disclosed in U.S. Pat. No.3,914,250, WO00/77010, WO02/36596, WO02/48124, WO02/10169, WO01/66548,WO02/44152, WO02/51844, WO02/40456, and WO02/40457; 5HT6 receptormodulators, such as those in WO03/030901, WO03/035061, WO03/039547, andthe like; acyl-estrogens, such as oleoyl-estrone, disclosed in delMar-Grasa, M. et al, Obesity Research, 9:202-9 (2001) and JapanesePatent Application No. JP 2000256190; anorectic bicyclic compounds suchas 1426 (Aventis) and 1954 (Aventis), and the compounds disclosed inWO00/18749, WO01/32638, WO01/62746, WO01/62747, and WO03/015769; CB 1(cannabinoid-1 receptor) antagonist/inverse agonists such as rimonabant(Acomplia; Sanofi), SR-147778 (Sanofi), SR-141716 (Sanofi), BAY 65-2520(Bayer), and SLV 319 (Solvay), and those disclosed in patentpublications U.S. Pat. Nos. 4,973,587, 5,013,837, 5,081,122, U.S. Pat.Nos. 5,112,820, 5,292,736, 5,532,237, 5,624,941, 6,028,084, 6,509,367,6,509,367, WO96/33159, WO97/29079, WO98/31227, WO98/33765, WO98/37061,WO98/41519, WO98/43635, WO98/43636, WO99/02499, WO00/10967, WO00/10968,WO01/09120, WO01/58869, WO01/64632, WO01/64633, WO01/64634, WO01/70700,WO01/96330, WO02/076949, WO03/006007, WO03/007887, WO03/020217,WO03/026647, WO03/026648, WO03/027069, WO03/027076, WO03/027114,WO03/037332, WO03/040107, WO03/086940, WO03/084943 and EP658546; CCK-A(cholecystokinin-A) agonists, such as AR-R 15849, GI 181771 (GSK),JMV-180, A-71378, A-71623 and SR146131 (Sanofi), and those described inU.S. Pat. No. 5,739,106; CNTF (Ciliary neurotrophic factors), such asGI-181771 (Glaxo-SmithKline), SRI 46131 (Sanofi Synthelabo),butabindide, PD 170,292, and PD 149164 (Pfizer); CNTF derivatives, suchas Axokine® (Regeneron), and those disclosed in WO94/09134, WO98/22128,and WO99/43813; dipeptidyl peptidase IV (DP-IV) inhibitors, such asisoleucine thiazolidide, valine pyrrolidide, NVP-DPP728, LAF237, P93/01,P 3298, TSL 225 (tryptophyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylicacid; disclosed by Yamada et al, Bioorg. & Med. Chem. Lett. 8 (1998)1537-1540), TMC-2A/2B/2C, CD26 inhibtors, FE 999011, P9310/K364, VIP0177, SDZ 274-444, 2-cyanopyrrolidides and 4-cyanopyrrolidides asdisclosed by Ashworth et al, Bioorg. & Med. Chem. Lett., Vol. 6, No. 22,pp 1163-1166 and 2745-2748 (1996) and the compounds disclosed patentpublications. WO99/38501, WO99/46272, WO99/67279 (Probiodrug),WO99/67278 (Probiodrug), WO99/61431 (Probiodrug), WO02/083128,WO02/062764, WO03/000180, WO03/000181, WO03/000250, WO03/002530,WO03/002531, WO03/002553, WO03/002593, WO03/004498, WO03/004496,WO03/017936, WO03/024942, WO03/024965, WO03/033524, WO03/037327 andEP1258476; growth hormone secretagogue receptor agonists/antagonists,such as NN703, hexarelin, MK-0677 (Merck), SM-130686, CP-424391(Pfizer), LY 444,711 (Eli Lilly), L-692,429 and L-163,255, and such asthose disclosed in U.S. Ser. No. 09/662,448, U.S. provisionalapplication 60/203,335, U.S. Pat. No. 6,358,951, US2002049196,US2002/022637, WO01/56592 and WO02/32888; H3 (histamine H3)antagonist/inverse agonists, such as thioperamide,3-(1H-imidazol-4-yl)propyl N-(4-pentenyl)carbamate), clobenpropit,iodophenpropit, imoproxifan, GT2394 (Gliatech), and A331440,O-[3-(1H-imidazol-4-yl)propanol]carbamates (Kiec-Kononowicz, K. et al.,Pharmazie, 55:349-55 (2000)), piperidine-containing histamineH3-receptor antagonists (Lazewska, D. et al., Pharmazie, 56:927-32(2001), benzophenone derivatives and related compounds (Sasse, A. etal., Arch. Pharm.(Weinheim) 334:45-52 (2001)), substitutedN-phenylcarbamates (Reidemeister, S. et al., Pharmazie, 55:83-6 (2000)),and proxifan derivatives (Sasse, A. et al., J. Med. Chem. 43:3335-43(2000)) and histamine H3 receptor modulators such as those disclosed inWO02/15905, WO03/024928 and WO03/024929; leptin derivatives, such asthose disclosed in U.S. Pat. Nos. 5,552,524, 5,552,523, 5,552,522,5,521,283, WO96/23513, WO96/23514, WO96/23515, WO96/23516, WO96/23517,WO96/23518, WO96/23519, and WO96/23520; leptin, including recombinanthuman leptin (PEG-OB, Hoffman La Roche) and recombinant methionyl humanleptin (Amgen); lipase inhibitors, such as tetrahydrolipstatin(orlistat/Xenical®), Triton WRl 339, RHC80267, lipstatin, teasaponin,diethylumbelliferyl phosphate, FL-386, WAY-121898, Bay-N-3176,valilactone, esteracin, ebelactone A, ebelactone B, and RHC 80267, andthose disclosed in patent publications WO01/77094, U.S. Pat. Nos.4,598,089, 4,452,813, USUS5512565, U.S. Pat. Nos. 5,391,571, 5,602,151,4,405,644, 4,189,438, and 4,242,453; lipid metabolism modulators such asmaslinic acid, erythrodiol, ursolic acid uvaol, betulinic acid, betulin,and the like and compounds disclosed in WO03/011267; Mc4r (melanocortin4 receptor) agonists, such as CHIR86036 (Chiron), ME-10142, ME-10145,and HS-131 (Melacure), and those disclosed in PCT publication Nos.WO99/64002, WO00/74679, WOO 1/991752, WOO 1/25192, WOO 1/52880, WOO1/74844, WOO 1/70708, WO01/70337, WO01/91752, WO02/059095, WO02/059107,WO02/059108, WO02/059117, WO02/06276, WO02/12166, WO02/11715,WO02/12178, WO02/15909, WO02/38544, WO02/068387, WO02/068388,WO02/067869, WO02/081430, WO03/06604, WO03/007949, WO03/009847,WO03/009850, WO03/013509, and WO03/031410; Mc5r (melanocortin 5receptor) modulators, such as those disclosed in WO97/19952, WO00/15826,WO00/15790, US20030092041; melanin-concentrating hormone 1 receptor(MCHR) antagonists, such as T-226296 (Takeda), SB 568849, SNP-7941(Synaptic), and those disclosed in patent publications WOO 1/21169,WO01/82925, WO01/87834, WO02/051809, WO02/06245, WO02/076929,WO02/076947, WO02/04433, WO02/51809, WO02/083134, WO02/094799,WO03/004027, WO03/13574, WO03/15769, WO03/028641, WO03/035624,WO03/033476, WO03/033480, JP13226269, and JP1437059; mGluR5 modulatorssuch as those disclosed in WO03/029210, WO03/047581, WO03/048137,WO03/051315, WO03/051833, WO03/053922, WO03/059904, and the like;serotoninergic agents, such as fenfluramine (such as Pondimin®(Benzeneethanamine, N-ethyl-alpha-methyl-3-(trifluoromethyl)-,hydrochloride), Robbins), dexfenfluramine (such as Redux®(Benzeneethanamine, N-ethyl-alpha-methyl-3-(trifluoromethyl)-,hydrochloride), Interneuron) and sibutramine ((Meridia®,Knoll/Reductil™) including racemic mixtures, as optically pure isomers(+) and (−), and pharmaceutically acceptable salts, solvents, hydrates,clathrates and prodrugs thereof including sibutramine hydrochloridemonohydrate salts thereof, and those compounds disclosed in U.S. Pat.Nos. 4,746,680, 4,806,570, and 5,436,272, US20020006964, WOO 1/27068,and WOO 1/62341; NE (norepinephrine) transport inhibitors, such as GW320659, despiramine, talsupram, and nomifensine; NPY 1 antagonists, suchas BIBP3226, J-115814, BIBO 3304, LY-357897, CP-671906, GI-264879A, andthose disclosed in U.S. Pat. No. 6,001,836, WO96/14307, WO01/23387,WO99/51600, WO01/85690, WO01/85098, WO01/85173, and WO01/89528; NPY5(neuropeptide Y Y5) antagonists, such as 152,804, GW-569180A,GW-594884A, GW-587081X, GW-548118X, FR235208, FR226928, FR240662,FR252384, 1229U91, GI-264879A, CGP71683A, LY-377897, LY-366377,PD-160170, SR-120562A, SR-120819A, JCF-104, and H409/22 and thosecompounds disclosed in patent publications U.S. Pat. Nos. 6,140,354,6,191,160, 6,218,408, 6,258,837, 6,313,298, 6,326,375, 6,329,395,6,335,345, 6,337,332, 6,329,395, 6,340,683, EP01010691, EP-01044970,WO97/19682, WO97/20820, WO97/20821, WO97/20822, WO97/20823, WO98/27063,WO00/107409, WO00/185714, WO00/185730, WO00/64880, WO00/68197,WO00/69849, WO/0113917, WO01/09120, WO01/14376, WO01/85714, WO01/85730,WO01/07409, WO01/02379, WO01/23388, WO01/23389, WOO 1/44201, WO01/62737,WO01/62738, WO01/09120, WO02/20488, WO02/22592, WO02/48152, WO02/49648,WO02/051806, WO02/094789, WO03/009845, WO03/014083, WO03/022849,WO03/028726 and Norman et al, J. Med. Chem. 43:4288-4312 (2000); opioidantagonists, such as nalmefene (REVEX®), 3-methoxynaltrexone,methylnaltrexone, naloxone, and naltrexone (e.g. PT901; PainTherapeutics, Inc.) and those disclosed in US20050004155 and WO00/21509;orexin antagonists, such as SB-334867-A and those disclosed in patentpublications WO01/96302, WO01/68609, WO02/44172, WO02/51232, WO02/51838,WO02/089800, WO02/090355, WO03/023561, WO03/032991, and WO03/037847; PDEinhibitors (e.g. compounds which slow the degradation of cyclic AMP(cAMP) and/or cyclic GMP (cGMP) by inhibition of the phosphodiesterases,which can lead to a relative increase in the intracellular concentrationof cAMP and cGMP; possible PDE inhibitors are primarily those substanceswhich are to be numbered among the class consisting of the PDE3inhibitors, the class consisting of the PDE4 inhibitors and/or the classconsisting of the PDE5 inhibitors, in particular those substances whichcan be designated as mixed types of PDE3/4 inhibitors or as mixed typesof PDE3/4/5 inhibitors) such as those disclosed in patent publicationsDE1470341, DE2108438, DE2123328, DE2305339, DE2305575, DE2315801,DE2402908, DE2413935, DE2451417, DE2459090, DE2646469, DE2727481,DE2825048, DE2837161, DE2845220, DE2847621, DE2934747, DE3021792,DE3038166, DE3044568, EP000718, EP0008408, EP0010759, EP0059948,EP0075436, EP0096517, EPOl 12987, EPOl 16948, EP0150937, EP0158380,EP0161632, EP0161918, EP0167121, EP0199127, EP0220044, EP0247725,EP0258191, EP0272910, EP0272914, EP0294647, EP0300726, EP0335386,EP0357788, EP0389282, EP0406958, EP0426180, EP0428302, EP0435811,EP0470805, EP0482208, EP0490823, EP0506194, EP0511865, EP0527117,EP0626939, EP0664289, EP0671389, EP0685474, EP0685475, EP0685479,JP92234389, JP94329652, JP95010875, U.S. Pat. Nos. 4,963,561, 5,141,931,WO9117991, WO9200968, WO9212961, WO9307146, WO9315044, WO9315045,WO9318024, WO9319068, WO9319720, WO9319747, WO9319749, WO9319751,WO9325517, WO9402465, WO9406423, WO9412461, WO9420455, WO9422852,WO9425437, WO9427947, WO9500516, WO9501980, WO9503794, WO9504045,WO9504046, WO9505386, WO9508534, WO9509623, WO9509624, WO9509627,WO9509836, WO9514667, WO9514680, WO9514681, WO9517392, WO9517399,WO9519362, WO9522520, WO9524381, WO9527692, WO9528926, WO9535281,WO9535282, WO9600218, WO9601825, WO9602541, WO9611917, DE3142982, DEl116676, DE2162096, EP0293063, EP0463756, EP0482208, EP0579496, EP0667345U.S. Pat. No. 6,331,543, US20050004222 (including those disclosed informulas I-XIII and paragraphs 37-39, 85-0545 and 557-577), WO9307124,EP0163965, EP0393500, EP0510562, EP0553174, WO9501338 and WO9603399, aswell as PDE5 inhibitors (such as RX-RA-69, SCH-51866, KT-734,vesnarinone, zaprinast, SKF-96231, ER-21355, BF/GP-385, NM-702 andsildenafil (Viagra™)), PDE4 inhibitors (such as etazolate, ICI63197,RP73401, imazolidinone (RO-20-1724), MEM 1414 (R1533/R1500; PharmaciaRoche), denbufylline, rolipram, oxagrelate, nitraquazone, Y-590,DH-6471, SKF-94120, motapizone, lixazinone, indolidan, olprinone,atizoram, KS-506-G, dipamfylline, BMY-43351, atizoram, arofylline,filaminast, PDB-093, UCB-29646, CDP-840, SKF-107806, piclamilast,RS-17597, RS-25344-000, SB-207499, TIBENELAST, SB-210667, SB-211572,SB-211600, SB-212066, SB-212179, GW-3600, CDP-840, mopidamol,anagrelide, ibudilast, amrinone, pimobendan, cilostazol, quazinone andN-(3,5-dichloropyrid-4-yl)-3-cyclopropylmethoxy4-difluoromethoxybenzamide,PDE3 inhibitors (such as ICI153, 100, bemorandane (RWJ 22867), MCI-154,UD-CG 212, sulmazole, ampizone, cilostamide, carbazeran, piroximone,imazodan, CI-930, siguazodan, adibendan, saterinone, SKF-95654,SDZ-MKS-492, 349-U-85, emoradan, EMD-53998, EMD-57033, NSP-306, NSP-307,revizinone, NM-702, WIN-62582 and WIN-63291, enoximone and milrinone,PDE3/4 inhibitors (such as benafentrine, trequinsin, ORG-30029,zardaverine, L-686398, SDZ-ISQ-844, ORG-20241, EMD-54622, andtolafentrine) and other PDE inhibitors (such as vinpocetin, papaverine,enprofylline, cilomilast, fenoximone, pentoxifylline, roflumilast,tadalafil (Cialis®), theophylline, and vardenafil (Levitra®);Neuropeptide Y2 (NPY2) agonists include but are not limited to:polypeptide YY and fragments and variants thereof (e.g. YY3-36(PYY3-36)(N. Engl. J. Med. 349:941, 2003; IKPEAPGE DASPEELNRY YASLRHYLNLVTRQRY (SEQ ID NO:260)) and PYY agonists such as those disclosed inWO02/47712, WO03/026591, WO03/057235, and WO03/027637; serotoninreuptake inhibitors, such as, paroxetine, fluoxetine (Prozac™),fluvoxamine, sertraline, citalopram, and imipramine, and those disclosedin U.S. Pat. Nos. 6,162,805, 6,365,633, WO03/00663, WOO 1/27060, and WOO1/162341; thyroid hormone β agonists, such as KB-2611 (KaroBioBMS), andthose disclosed in WO02/15845, WO97/21993, WO99/00353, GB98/284425, U.S.Provisional Application No. 60/183,223, and Japanese Patent ApplicationNo. JP 2000256190; UCP-I (uncoupling protein-1), 2, or 3 activators,such as phytanic acid,4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-napthalenyl)-1-propenyl]benzoicacid (TTNPB), retinoic acid, and those disclosed in WO99/00123; (33(beta adrenergic receptor 3) agonists, such as AJ9677/TAK677(Dainippon/Takeda), L750355 (Merck), CP331648 (Pfizer), CL-316,243, SB418790, BRL-37344, L-796568, BMS-196085, BRL-35135A, CGP12177A, BTA-243,GW 427353, Trecadrine, Zeneca D7114, N-5984 (Nisshin Kyorin), LY-377604(Lilly), SR 59119A, and those disclosed in U.S. Pat. Nos. 5,541,204,5,770,615, 5,491,134, 5,776,983, 488,064, 5,705,515, 5,451,677,WO94/18161, WO95/29159, WO97/46556, WO98/04526 and WO98/32753,WO01/74782, WO02/32897, WO03/014113, WO03/016276, WO03/016307,WO03/024948, WO03/024953 and WO03/037881; noradrenergic agentsincluding, but not limited to, diethylpropion (such as Tenuate®(1-propanone, 2-(diethylamino)-1-phenyl-, hydrochloride), Merrell),dextroamphetamine (also known as dextroamphetamine sulfate,dexamphetamine, dexedrine, Dexampex, Ferndex, Oxydess II, Robese,Spancap #1), mazindol ((or5-(p-chlorophenyl)-2,5-dihydro-3H-imidazo[2,1-a]isoindol-5-01) such asSanorex®, Novartis or Mazanor®, Wyeth Ayerst), phenylpropanolamine (orBenzenemethanol, alpha-(1-aminoethyl)-, hydrochloride), phentermine ((orPhenol, 3-[[4,5-duhydro-1H-imidazol-2-yl)ethyl](4-methylpheny-1)amino],monohydrochloride) such as Adipex-P®, Lemmon, FASTIN®, Smith-KlineBeecham and Ionamin®, Medeva), phendimetrazine ((or(2S,3S)-3,4-Dimethyl-2phenylmorpholine L-(+)-tartrate (1:1)) such asMetra® (Forest), Plegine® (Wyeth-Ay erst), Prelu-2® (BoehringerIngelheim), and Statobex® (Lemmon), phendamine tartrate (such asThephorin®(2,3,4,9-Tetrahydro-2-methyl-9-phenyl-1H-indenol[2,1-c]pyridineL-(+)-tartrate (1:1)), Hoffmann-LaRoche), methamphetamine (such asDesoxyn®, Abbot ((S)—N, (alpha)-dimethylbenzeneethanaminehydrochloride)), and phendimetrazine tartrate (such as Bontril®Slow-Release Capsules, Amarin (-3,4-Dimethyl-2-phenylmorpholineTartrate); fatty acid oxidation upregulator/inducers such as Famoxin®(Genset); monamine oxidase inhibitors including but not limited tobefloxatone, moclobemide, brofaromine, phenoxathine, esuprone, befol,toloxatone, pirlindol, amiflamine, sercloremine, bazinaprine,lazabemide, milacemide, caroxazone and other certain compounds asdisclosed by WO01/12176; and other anti-obesity agents such as 5HT-2agonists, ACC (acetyl-CoA carboxylase) inhibitors such as thosedescribed in WO03/072197, alpha-lipoic acid (alpha-LA), A0D9604,appetite suppressants such as those in WO03/40107, ATL-962 (AlizymePLC), benzocaine, benzphetamine hydrochloride (Didrex), bladderwrack(focus vesiculosus), BRS3 (bombesin receptor subtype 3) agonists,bupropion, caffeine, CCK agonists, chitosan, chromium, conjugatedlinoleic acid, corticotropin-releasing hormone agonists,dehydroepiandrosterone, DGAT1 (diacylglycerol acyltransferase 1)inhibitors, DGAT2 (diacylglycerol acyltransferase 2) inhibitors,dicarboxylate transporter inhibitors, ephedra, exendin-4 (an inhibitorof glp-1) FAS (fatty acid synthase) inhibitors (such as Cerulenin andC75), fat resorption inhibitors (such as those in WO03/053451, and thelike), fatty acid transporter inhibitors, natural water soluble fibers(such as psyllium, plantago, guar, oat, pectin), galanin antagonists,galega (Goat's Rue, French Lilac), garcinia cambogia, germander(teucrium chamaedrys), ghrelin antibodies and ghrelin antagonists (suchas those disclosed in WO01/87335, and WO02/08250), polypeptide hormonesand variants thereof which affect the islet cell secretion, such as thehormones of the secretin/gastric inhibitory polypeptide (GIP)/vasoactiveintestinal polypeptide (VIP)/pituitary adenylate cyclase activatingpolypeptide (PACAP)/glucagon-like polypeptide II(GLP-II)/glicentin/glucagon gene family and/or those of theadrenomedullin/amylin/calcitonin gene related polypeptide (CGRP) genefamily includingGLP-1 (glucagon-like polypeptide 1) agonists (e.g. (1)exendin-4, (2) those GLP-I molecules described in US20050130891including GLP-1(7-34), GLP-1(7-35), GLP-1(7-36) or GLP-1(7-37) in itsC-terminally carboxylated or amidated form or as modified GLP-Ipolypeptides and modifications thereof including those described inparagraphs 17-44 of US20050130891, and derivatives derived fromGLP-1-(7-34)COOH and the corresponding acid amide are employed whichhave the following general formula:R—NH-HAEGTFTSDVSYLEGQAAKEFIAWLVK-CONH₂ (SEQ ID NO: 261) wherein R═H oran organic compound having from 1 to 10 carbon atoms. Preferably, R isthe residue of a carboxylic acid. Particularly preferred are thefollowing carboxylic acid residues: formyl, acetyl, propionyl,isopropionyl, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl,tert-butyl.) and glp-1 (glucagon-like polypeptide-1), glucocorticoidantagonists, glucose transporter inhibitors, growth hormonesecretagogues (such as those disclosed and specifically described inU.S. Pat. No. 5,536,716), interleukin-6 (IL-6) and modulators thereof(as in WO03/057237, and the like), L-carnitine, Mc3r (melanocortin 3receptor) agonists, MCH2R (melanin concentrating hormone 2R)agonist/antagonists, melanin concentrating hormone antagonists,melanocortin agonists (such as Melanotan II or those described in WO99/64002 and WO 00/74679), nomame herba, phosphate transporterinhibitors, phytopharm compound 57 (CP 644,673), pyruvate, SCD-I(stearoyl-CoA desaturase-1) inhibitors, T71 (Tularik, Inc., BoulderColo.), Topiramate (Topimax®, indicated as an anti-convulsant which hasbeen shown to increase weight loss), transcription factor modulators(such as those disclosed in WO03/026576), β-hydroxy steroiddehydrogenase-1 inhibitors (β-HSD-I), β-hydroxy-β-methylbutyrate, p57(Pfizer), Zonisamide (Zonegran™, indicated as an anti-epileptic whichhas been shown to lead to weight loss), and the agents disclosed inUS20030119428 paragraphs 20-26.

1.3.1.7 Phosphodiesterase Inhibitors

In certain embodiments, the regimen of combination therapy includes theadministration of one or more phosphodiesterase (“PDE”) inhibitors. PDEinhibitors slow the degradation of cyclic AMP (cAMP) and/or cyclic GMP(cGMP) by inhibiting phosphodiesterases, which can lead to a relativeincrease in the intracellular concentration of cAMP and/or cGMP.Non-limiting examples of PDE inhibitors that can be used in combinationwith the GCC agonists of the invention include PDE3 inhibitors, PDE4inhibitors and/or PDE5 inhibitors, in particular those substances whichcan be designated as mixed types of PDE3/4 inhibitors or as mixed typesof PDE3/4/5 inhibitors. Non-limiting examples of such PDE inhibitors aredescribed in the following patent applications and patents: DE1470341,DE2108438, DE2123328, DE2305339, DE2305575, DE2315801, DE2402908,DE2413935, DE2451417, DE2459090, DE2646469, DE2727481, DE2825048,DE2837161, DE2845220, DE2847621, DE2934747, DE3021792, DE3038166,DE3044568, EP000718, EP0008408, EP0010759, EP0059948, EP0075436,EP0096517, EPOl 12987, EPOl 16948, EP0150937, EP0158380, EP0161632,EP0161918, EP0167121, EP0199127, EP0220044, EP0247725, EP0258191,EP0272910, EP0272914, EP0294647, EP0300726, EP0335386, EP0357788,EP0389282, EP0406958, EP0426180, EP0428302, EP0435811, EP0470805,EP0482208, EP0490823, EP0506194, EP0511865, EP0527117, EP0626939,EP0664289, EP0671389, EP0685474, EP0685475, EP0685479, JP92234389,JP94329652, JP95010875, U.S. Pat. Nos. 4,963,561, 5,141,931, WO9117991,WO9200968, WO9212961, WO9307146, WO9315044, WO9315045, WO9318024,WO9319068, WO9319720, WO9319747, WO9319749, WO9319751, WO9325517,WO9402465, WO9406423, WO9412461, WO9420455, WO9422852, WO9425437,WO9427947, WO9500516, WO9501980, WO9503794, WO9504045, WO9504046,WO9505386, WO9508534, WO9509623, WO9509624, WO9509627, WO9509836,WO9514667, WO9514680, WO9514681, WO9517392, WO9517399, WO9519362,WO9522520, WO9524381, WO9527692, WO9528926, WO9535281, WO9535282,WO9600218, WO9601825, WO9602541, WO9611917, DE3142982, DEl 116676,DE2162096, EP0293063, EP0463756, EP0482208, EP0579496, EP0667345 U.S.Pat. No. 6,331,543, US20050004222 (including those disclosed in formulasI—XIII and paragraphs 37-39, 85-0545 and 557-577) and WO9307124,EP0163965, EP0393500, EP0510562, EP0553174, WO9501338 and WO9603399.PDE5 inhibitors which may be mentioned by way of example are RX-RA-69,SCH-51866, KT-734, vesnarinone, zaprinast, SKF-96231, ER-21355,BF/GP-385, NM-702 and sildenafil (Viagra®). PDE4 inhibitors which may bementioned by way of example are RO-20-1724, MEM 1414 (R1533/R1500;Pharmacia Roche), DENBUFYLLINE, ROLIPRAM, OXAGRELATE, NITRAQUAZONE,Y-590, DH-6471, SKF-94120, MOTAPIZONE, LIXAZINONE, INDOLIDAN, OLPRINONE,ATIZORAM, KS-506-G, DIPAMFYLLINE, BMY-43351, ATIZORAM, AROFYLLINE,FILAMINAST, PDB-093, UCB-29646, CDP-840, SKF-107806, PICLAMILAST,RS-17597, RS-25344-000, SB-207499, TIBENELAST, SB-210667, SB-211572,SB-211600, SB-212066, SB-212179, GW-3600, CDP-840, MOPIDAMOL,ANAGRELIDE, IBUDILAST, AMRINONE, PIMOBENDAN, CILOSTAZOL, QUAZINONE andN-(3,5-dichloropyrid-4-yl)-3-cyclopropylmethoxy4-difluoromethoxybenzamide.PDE3 inhibitors which may be mentioned by way of example are SULMAZOLE,AMPIZONE, CILOSTAMIDE, CARBAZERAN, PIROXIMONE, IMAZODAN, CI-930,SIGUAZODAN, ADIBENDAN, SATERINONE, SKF-95654, SDZ-MKS-492, 349-U-85,EMORADAN, EMD-53998, EMD-57033, NSP-306, NSP-307, REVIZINONE, NM-702,WIN-62582 and WIN-63291, ENOXIMONE and MILRINONE. PDE3/4 inhibitorswhich may be mentioned by way of example are BENAFENTRINE, TREQUINSIN,ORG-30029, ZARDAVERINE, L-686398, SDZ-ISQ-844, ORG-20241, EMD-54622, andTOLAFENTRINE. Other PDE inhibitors include: cilomilast, pentoxifylline,roflumilast, tadalafil (Cialis®), theophylline, and vardenafil(Levitra®), zaprinast (PDE5 specific). GCC AGONIST

1.3.1.8 Analgesic Agents

In certain embodiments, the regimen of combination therapy includes theadministration of one or more analgesic agents, e.g., an analgesiccompound or an analgesic polypeptide. In some embodiments, the GCCagonist formulation is administered simultaneously or sequentially withone or more analgesic agents. In other embodiments, the GCC agonist iscovalently linked or attached to an analgesic agent to create atherapeutic conjugate. Non-limiting examples of analgesic agents thatcan be used include calcium channel blockers, 5HT receptor antagonists(for example 5HT3, 5HT4 and 5HT1 receptor antagonists), opioid receptoragonists (loperamide, fedotozine, and fentanyl), NK1 receptorantagonists, CCK receptor agonists (e.g., loxiglumide), NK1 receptorantagonists, NK3 receptor antagonists, norepinephrine-serotonin reuptakeinhibitors (NSRI), vanilloid and cannabanoid receptor agonists, andsialorphin. Further examples of analgesic agents in the various classesare known in the art.

In one embodiment, the analgesic agent is an analgesic polypeptideselected from the group consisting of sialorphin-related polypeptides,including those comprising the amino acid sequence QHNPR (SEQ ID NO:239), including: VQHNPR (SEQ ID NO: 240); VRQHNPR (SEQ ID NO: 241);VRGQHNPR (SEQ ID NO: 242); VRGPQHNPR (SEQ ID NO: 243); VRGPRQHNPR (SEQID NO: 244); VRGPRRQHNPR (SEQ ID NO: 245); and RQHNPR (SEQ ID NO: 246).Sialorphin-related polypeptides bind to neprilysin and inhibitneprilysin-mediated breakdown of substance P and Met-enkephalin. Thus,compounds or polypeptides that are inhibitors of neprilysin are usefulanalgesic agents which can be administered with the GCC agonistsdescribed herein or covalently linked to a GCC agonist to form atherapeutic conjugate. Sialorphin and related polypeptides are describedin U.S. Pat. No. 6,589,750; U.S. 20030078200 A1; and WO 02/051435 A2.

In another embodiment, a GCC agonist formulation of the invention isadministered as part of a regimen of combination therapy with an opioidreceptor antagonist or agonist. In one embodiment, the GCC agonist andthe opioid receptor antagonist or agonist are linked via a covalentbond. Non-limiting examples of opioid receptor antagonists includenaloxone, naltrexone, methyl nalozone, nalmefene, cypridime, betafunaltrexamine, naloxonazine, naltrindole, nor-binaltorphimine,enkephalin pentapeptide (HOE825; Tyr-D-Lys-Gly-Phe-L-homoserine),trimebutine, vasoactive intestinal polypeptide, gastrin, glucagons.Non-limiting examples of opioid receptor agonists include fedotozine,asimadoline, and ketocyclazocine, the compounds described in WO03/097051and WO05/007626, morphine, diphenyloxylate, frakefamide(H-Tyr-D-Ala-Phe(F)-Phe-NH 2; WO 01/019849 A1), and loperamide.

Further non-limiting examples of analgesic agents that can be used in aregimen of combination therapy along with the GCC agonist formulationsof the invention include the dipeptide Tyr-Arg (kyotorphin); thechromogranin-derived polypeptide (CgA 47-66; See, e.g., Ghia et al. 2004Regulatory polypeptides 119:199); CCK receptor agonists such ascaerulein; conotoxin polypeptides; peptide analogs of thymulin (FRApplication 2830451); CCK (CCKa or CCKb) receptor antagonists, includingloxiglumide and dexloxiglumide (the R-isomer of loxiglumide) (WO88/05774); 5-HT4 agonists such as tegaserod (Zelnorm®), mosapride,metoclopramide, zacopride, cisapride, renzapride, benzimidazolonederivatives such as BIMU 1 and BIMU 8, and lirexapride; calcium channelblockers such as ziconotide and related compounds described in, forexample, EP625162B1, U.S. Pat. Nos. 5,364,842, 5,587,454, 5,824,645,5,859,186, 5,994,305, 6,087,091, 6,136,786, WO 93/13128 A1, EP 1336409A1, EP 835126 A1, EP 835126 B1, U.S. Pat. Nos. 5,795,864, 5,891,849,6,054,429, WO 97/01351 A1; NK-I, receptor antagonists such as aprepitant(Merck & Co Inc), vofopitant, ezlopitant (Pfizer, Inc.), R-673(Hoffmann-La Roche Ltd), SR-48968 (Sanofi Synthelabo), CP-122,721(Pfizer, Inc.), GW679769 (Glaxo Smith Kline), TAK-637 (Takeda/Abbot),SR-14033, and related compounds described in, for example, EP 873753 A1,US 20010006972 A1, US 20030109417 A1, WO 01/52844 A1 (for a review seeGiardina et al. 2003. Drugs 6:758); NK-2 receptor antagonists such asnepadutant (Menarini Ricerche SpA), saredutant (Sanoft-Synthelabo),GW597599 (Glaxo Smith Kline), SR-144190 (Sanofi-Synthelabo) andUK-290795 (Pfizer Inc); NK3 receptor antagonists such as osanetant(SR-142801; Sanoft-Synthelabo), SSR-241586, talnetant and relatedcompounds described in, for example, WO 02/094187 A2, EP 876347 A1, WO97/21680 A1, U.S. Pat. No. 6,277,862, WO 98/1 1090, WO 95/28418, WO97/19927, and Boden et al. (J Med Chem. 39:1664-75, 1996);norepinephrine-serotonin reuptake inhibitors (NSRI) such as milnacipranand related compounds described in WO 03/077897; and vanilloid receptorantagonists such as arvanil and related compouds described in WO01/64212 A1.

In addition to sialorphin-related polypeptides, analgesic polypeptidesinclude: AspPhe, endomorphin-1, endomorphin-2, nocistatin, dalargin,lupron, ziconotide, and substance P.

EXAMPLES Example 1: Synthesis and Purification of GCC Agonist Peptides

The GCC agonist peptides were synthesized using standard methods forsolid-phase peptide synthesis. Either a Boc/Bzl or Fmoc/tBu protectinggroup strategy was seleceted depending upon the scale of the peptide tobe produced. In the case of smaller quantities, it is possible to getthe desired product using an Fmoc/tBu protocol, but for largerquantities (1 g or more), Boc/Bzl is superior.

In each case the GCC agonist peptide was started by either using apre-loaded Wang (Fmoc) or Merrifield (Boc) or Pam (Boc) resin. Forproducts with C-terminal Leu, Fmoc-Leu-Wang (D-1115) or Boc-Leu-Pamresin (D-1230) or Boc-Leu-Merrifield (D-1030) Thus, for peptidescontaining the C-terminal d-Leu, the resin was Fmoc-dLeu-Wang Resin(D-2535) and Boc-dLeu-Merrifield, Boc-dLeu-Pam-Resin (Bachem ProductD-1230 and D-1590, respectively) (SP-332 and related analogs). Forpeptides produced as C-terminal amides, a resin with Ramage linker(Bachem Product D-2200) (Fmoc) or mBHA (Boc) (Bachem Product D-1210 wasused and loaded with the C-terminal residue as the first synthetic step.

Fmoc-tBu Overview

Each synthetic cycle consisted deprotection with 20% piperidine in DMF.Resin washes were accomplished with alternating DMF and IpOH to swelland shrink the resin, respectively. Peptide synthesis elongated thechain from the C-terminus to the N-terminus. Activation chemistry foreach amino acid was with HBTU/DIEA in a 4 fold excess for 45 minutes. Inautomated chemistries, each amino acid was double coupled to maximizethe coupling efficiency. To insure the correct position of disulfidebonds, the Cys residues were introduced as Cys (Acm) at positions 15 and7. Cys (Trt) was positioned at Cys4 and Cys12. This protecting groupstrategy yields the correct topoisomer as the dominant product (75:25).(For enterotoxin analogs, a third disulfide bond protecting group (Mob)was utilized).

For peptides containing C-terminal Aeea (aminoethyloxyethyloxyacetyl)groups, these were coupled to a Ramage amide linker using the sameactivation chemistry above by using an Fmoc-protected Aeea derivative.The Cys numbering in these cases remains the same and the positioning ofthe protecting groups as well. For the peptides containing theN-terminal extension of Aeea, the Cys residue numbering will beincreased by three Cys4 becomes Cys7, Cys12 becomes Cys15; Cys7 becomesCys10 and Cys 15 becomes Cys18. The latter pair is protected with Acmand the former pair keeps the Trt groups.

For analogs containing D-amino acid substitutions, these were introduceddirectly by incorporating the correctly protected derivative at thedesired position using the same activation chemistry described in thisdocument. For Fmoc strategies, Fmoc-dAsn(Trt)-OH, Fmoc-dAsn(Xan)-OH,Fmoc-dAsp(tBu)-OH, Fmoc-dGlu(tBu)-OH and for Boc strategies,Boc-dAsn(Xan)-OH, Boc-dAsn(Trt)-OH, Boc-dAsp(Chx), Boc-dAsp(Bzl)-OH,Boc-dGlu(Chx)-OH and Boc-dGlu(Bzl)-OH would be utilized.

Each peptide is cleaved from the solid-phase support using a cleavagecocktail of TFA:H2O:Trisisopropylsilane (8.5:0.75:0.75) ml/g of resinfor 2 hr at RT. The crude deprotected peptide is filtered to remove thespent resin beads and precipitated into ice-cold diethylether.

Each disulfide bonds was introduced orthogonally. Briefly, the crudesynthetic product was dissolved in water containing NH₄OH to increasethe pH to 9. Following complete solubilization of the product, thedisulfide bond was made between the Trt deprotected Cys residues bytitration with H₂O₂. The monocyclic product was purified by RP-HPLC. Thepurified mono-cyclic product was subsequently treated with a solution ofiodine to simultaneously remove the Acm protecting groups and introducethe second disulfide bond.

For enterotoxin analogs, the Mob group was removed via treatment of thedicyclic product with TFA 85% containing 10% DMSO and 5% thioanisole for2 hr at RT.

Each product was then purified by RP-HPLC using a combination buffersystem of TEAP in H2O versus MeCN, followed by TFA in H2O versus MeCN.Highly pure fractions were combined and lyophilized. The final productwas converted to an Acetate salt using either ion exchange with Acetateloaded Dow-Ex resin or using RP-HPLC using a base-wash step with NH₄OAcfollowed by 1% AcOH in water versus MeCN.

It is also possible to prepare enterotoxin analogs using a randomoxidation methodology using Cys(Trt) in Fmoc or Cys(MeB) in Boc.Following cleavage, the disulfide bonds can be formed using disulfideinterchange redox pairs such as glutathione (red/ox) and/orcysteine/cystine. This process will yield a folded product that thedisulfide pairs must be determined as there would be no way of knowingtheir position directly.

Boc-Bzl Process

Peptide synthesis is initiated on a Merrifield or Pam pre-loaded resinor with mBHA for peptides produced as C-terminal amides. Each syntheticcycle consists of a deprotection step with 50% TFA in MeCL2. The resinis washed repetitively with MeCl2 and MeOH. The TFA salt formed isneutralized with a base wash of 10% TEA in MeCl2. The resin is washedwith MeCl2 and MeOH and lastly with DMF prior to coupling steps. Acolorimetric test is conducted to ensure deprotection. Each coupling ismediated with diisopropyl carbodiimide with HOBT to form the activeester. Each coupling is allowed to continue for 2 hr at RT or overnighton difficult couplings. Recouplings are conducted with either Uronium orPhosphonium reagents until a negative colorimetric test is obtained forfree primary amines. The resin is then washed with DMF, MeCl2 and MeOHand prepared for the next solid-phase step. Cys protection utilizesCys(Acm) at positions 7 and 15, and Cys(MeB) at Cys 4 and Cys12.

Cleavage and simultaneous deprotection is accomplished by treatment withHF using anisole as a scavenger (9:1:1) ml:ml:g (resin) at 0° C. for 60min. The peptide is subsequently extracted from the resin andprecipitated in ice cold ether. The introduction of disulfide bonds andpurification follows the exact same protocol described above for theFmoc-produced product.

Example 2: In Vitro Biological and Chemical Stability of SP-304 afterIncubation in Simulated Gastric Fluid (SGF)

The stability of SP-304 in the presence of simulated gastric fluid (SGF)was determined by biological activity measurements and HPLC analyses(FIGS. 1A & 1B). SP-304 (final concentration of 8.5 mg/ml) was incubatedin SGF (Proteose peptone (8.3 g/liter; Difco), D-Glucose (3.5 g/liter;Sigma), NaCl (2.05 g/liter; Sigma), KH 2PO4 (0.6 g/liter; Sigma), CaCl₂)(0.11 g/liter), KCl (0.37 g/liter; Sigma), Porcine bile (final 1×concentration 0.05 g/liter; Sigma) in PBS, Lysozyme (final 1×concentration 0.10 g/liter; Sigma) in PBS, Pepsin (final 1×concentration 0.0133 g/liter; Sigma) in PBS). SGF was made on the day ofthe experiment and the pH was adjusted to 2.0±0.1 using HCl or NaOH asnecessary. After the pH adjustment, SGF is filter sterilized with 0.22μm membrane filters. SP-304 (final concentration of 8.5 mg/ml) wasincubated in SGF at 37° C. for 0, 15, 30, 45, 60 and 120 min,respectively, in triplicate aliquots. Following incubations, sampleswere snap frozen in dry ice then stored in a −80° C. freezer untilassayed in duplicate.

FIG. 1A shows a bar chart showing the biological activity of SP-304after incubation with SGF for times as indicated. The activity at 0 minwas taken as 100%. The data are an average of triplicates ±SD for eachdata point. The data demonstrate that SP-304 is resistant to breakdownin SGF for incubations lasting as long as two hours. In addition, thedata also suggest that the activity of SP-304 is unaltered by exposureto the acidic pH of the SGF.

The HPLC chromatograms of samples of SP-304 incubated in SGF for 0 and120 min are shown in FIG. 1B. Here, aliquots of the two samples wereanalyzed by HPLC using a previously developed method for analyzingSP-304 peptide. Samples from the SGF incubations were diluted to give afinal concentration 0.17 mg/mL of SP-304. The major peak of SP-304 didnot change following incubation with SGF, indicating that the peptidewas resistant to SGF treatment.

Example 3: In Vitro Biological and Chemical Stability of SP-304 afterIncubation in Simulated Intestinal Fluid (SIF)

The stability of SP-304 was also evaluated after incubation withsimulated intestinal fluid (SIF) by measuring its biological activityand by HPLC analyses (FIGS. 2A & 2B). SIF solution was prepared by themethod as described in the United States Pharmacopoeia, 24th edition,p2236. The recipe to prepare SIF solution was as described below. TheSIF solution contained NaCl (2.05 g/liter; Sigma), KH 2PO4 (0.6 g/liter;Sigma), CaCl₂) (0.11 g/liter), KCl (0.37 g/liter; Sigma), and Pacreatin10 mg/ml. The pH was adjusted to 6 and the solution was filtersterilized. A solution of SP-304 (8.5 mg/ml) was incubated in SGF at 37°C. for 0, 30, 60, 90, 120, 150 and 300 min respectively, in triplicatealiquots. Following incubations, samples were removed and snap frozenwith dry ice and stored in a −80° C. freezer until assayed in duplicate.FIG. 2A is a bar chart showing the ability of SP-304, after incubationin SIF for times as indicated, to stimulate cGMP synthesis in T84 cells.The cGMP stimulation activity at 0 min was taken as 100%. The data arean average of 3 triplicates ±SD. The data indicated that the biologicalactivity of SP-304 is reduced by about 30% following incubation in SIFfor 300 min.

The physical stability of SP-304 peptide exposed to SIF was evaluated byHPLC using the method described for SGF digestion. FIG. 2B shows HPLCchromatograms for SP-304 after incubation with heat-inactivated SIF for300 min, and SIF for 120 min, respectively. SP-304 treated withheat-inactivated SIF remained intact (Note: the major peak of SP-304eluting at 16.2 min), whereas SP-304 treated with SIF for 120 min wascompletely converted into another peak eluting at 9.4 min plus a fewminor additional peaks.

FIG. 3 is a schematic representation of the possible metabolites ofSP-304. The major degradation products involve Asn and Asp clipped fromthe N-terminus and Leu from the C-terminus of SP304. The fact that only30% reduction in biological activity was observed even after 2 hoursincubation in SIF implies that one or more of the degradation productsobserved in FIG. 2B are also biologically active. To address thispossibility, several truncated peptides were synthesized and evaluatedfor their abilities to stimulate cGMP synthesis in T84 cells (FIG. 4).

FIG. 4 shows data from the analyses of various peptides in the T84 cellcGMP stimulation assay (essentially as described in Shailubhai, et al.,Cancer Research 60, 5151-5157 (2000). Briefly, confluent monolayers ofT-84 cells in 24-well plates were washed twice with 250 μl of DMEMcontaining 50 mM HEPES (pH 7.4) and pre-incubated at 37° C. for 10minutes with 250 μl of DMEM containing 50 mM HEPES (pH 7.4) and 1 mMisobutyl methylxanthine (IBMX). Monolayers of T84 cells were thenincubated with 250 μl of DMEM containing 50 mM HEPES (pH 7.4) containingone of the peptides shown in the FIG. 4 at a concentration of 1.0 μM for30 min. After the 30 min incubation, the medium was aspirated and thereaction was terminated by the addition of 3% perchloric acid. Followingcentrifugation and the addition of NaOH (0.1 N) to neutralize the pH,intracellular cGMP levels were determined in lysates using a cGMP ELISAkit (Cat. No. 581021; Cayman Chemical, Ann Arbor, Mich.). Peptideincubations were run in duplicate, and samples taken from eachincubation were run as duplicates in the ELISA test.

The data indicate that SP-338, the 15-mer peptide missing the leucine(L) residue at the C-terminus of SP-304, retains about 80% of thebiological activity of the full length 16-mer SP-304 peptide. Thus, theC-terminal Leu clearly does make some contribution to the biologicalpotency of the peptide. Similarly, peptides SP-327, SP-329 and SP-331,which are all missing their C-terminal Leu, also showed a 20-25%reduction in biological potency relative to their counterpart parentpeptides SP-326, SP-328 and SP-330, respectively. In addition, the dataalso suggest that amino acid residues at the N-terminus may alsocontribute to the stability and/or potency of the peptides. Severaladditional peptides were synthesized with D-forms of amino acidsreplacing the corresponding L-forms at the C- and N-termini of thepeptides. These peptides were evaluated for their abilities to stimulatecGMP synthesis in T84 cells as shown in FIG. 5.

The results presented in FIG. 5 indicate that substitution of L-aminoacids with D-amino acids at the C- and N-termini did not significantlyalter their potency relative to SP-304. Peptides SP-332, SP-333 andSP-335 all showed comparable ability to stimulate cGMP synthesis in T84cells. These results suggest that the amino acid residues Asn, Asp andGlu at the N-terminus and Leu at the C-terminus can be replaced withtheir respective D-amino acid forms. On the other hand, substitution ofL-leucine with D-leucine at the 6^(th) position (SP-337) resulted invirtually complete loss of biological activity.

FIG. 7 (A-F) shows the stabilities of peptides SP-332, SP-333 and SP-304when incubated in SIF for two hours. The results demonstrate thatSP-333, which has D-Asn at the N-terminus and D-Leu at the C-terminus,remained virtually 100% biologically active after a two hour incubationin SIF (FIG. 7A), and remained virtually intact to digestion with SIFafter two hours (FIGS. 7F-1 & 7F-2). Subsequent incubation studies withSP-333 performed in SIF for up to 24 hours indicate that there is verylittle degradation even after 24 hours in SIF (FIG. 7G). The dataindicated that SP-333 is stable against digestion with SIF for up to 24hours. Peptide SP-332 with D-Leu at the C-terminus showed a minorreduction in potency following the 120 min incubation with SIF (FIG.7B). Interestingly, the HPLC analyses of SP-332 did not reveal anyclear-cut degradation of the peptide (FIGS. 7E-1 & 7E2), also suggestingthat this peptide is also almost completely resistant to proteoysis bySIF during the 2-hr incubation. On the other hand, peptide SP-304 lostabout 30% of its potency following digestion with SIF for just one hour(FIG. 7C). The HPLC analysis of SP-304 following SIF incubationconfirmed its degradation (FIGS. 7D-1 & 7D-2). These results suggestthat SP-304 undergoes substantial proteolysis following incubation withSIF within one hour.

Example 4: Cyclic GMP Stimulation Assays

The ability of the GCC agonist peptide to bind to and activate theintestinal GC-C receptor was tested using T84 human colon carcinoma cellline. Human T84 colon carcinoma cells were obtained from the AmericanType Culture Collection. Cells were grown in a 1:1 mixture of Ham's F-12medium and Dulbecco's modified Eagle's medium (DMEM) supplemented with10% fetal bovine serum, 100 U penicillin/ml, and 100 μg/ml streptomycin.The cells were fed fresh medium every third day and split at aconfluence of approximately 80%.

Biological activity of the GCC agonist peptides was assayed aspreviously reported (Shailubhai, et al., Cancer Research 60, 5151-5157(2000)). Briefly, the confluent monolayers of T-84 cells in 24-wellplates were washed twice with 250 μl of DMEM containing 50 mM HEPES (pH7.4), pre-incubated at 37° C. for 10 min with 250 μl of DMEM containing50 mM HEPES (pH 7.4) and 1 mM isobutylmethylxanthine (IBMX), followed byincubation with GCC agonist peptides (0.1 nM to 10.mu.M) for 30 min. Themedium was aspirated, and the reaction was terminated by the addition of3% perchloric acid. Following centrifugation, and neutralization with0.1 N NaOH, the supernatant was used directly for measurements of cGMPusing an ELISA kit (Caymen Chemical, Ann Arbor, Mich.).

FIG. 6 shows results from experiments evaluating the potency of peptides(via cGMP stimulation assay) having structures similar to the 14-merpeptide SP-339, also referred to as linaclotide. SP-339 is a truncatedanalog of the E. coli enterotoxin ST peptide. SP-354 was found to bevirtually identical to SP-339 in biological activity. Notably, peptideSP-353, which has a Ser residue at the 6^(th) position, was found to bemore potent than SP-339, and was the most potent of all the peptidestested. Peptide SP-355 which has a D-Tyr at the C-terminus showedconsiderably less potency than the other peptides tested.

Example 5: Peggylated Peptides

An additional strategy to render peptides more resistant towardsdigestion by digestive proteases is to peggylate them at the N- andC-terminus. The peptide SP-333 was peggylated with theaminoethyloxy-ethyloxy-acetic acid (Aeea) group at the C-terminus(SP-347) or at the N-terminus (SP-350) or at both termini (SP-343).Cyclic GMP synthesis in T84 cells was measured by the method asdescribed above.

The peptides SP-347 and SP-350 showed potencies comparable to SP-333 intheir abilities to stimulate cGMP synthesis in T84 cells. However,peptide SP-343 was considerably less potent as compared to the otherpeptides tested. The poor activity of SP-343 might be due to theconsiderable steric hindrance afforded by the large Aeea groups at bothtermini.

Example 6: Combination of Guanylate Cyclase Receptor Agonists withPhosphodiesterase Inhibitors

Regulation of intracellular concentrations of cyclic nucleotides (i.e.,cAMP and cGMP) and thus, signaling via these second messengers, has beengenerally considered to be governed by their rates of production versustheir rates of destruction within cells. Thus, levels of cGMP in tissuesand organs can also be regulated by the levels of expression ofcGMP-specific phosphodiesterases (cGMP-PDE), which are generallyoverexpressed in cancer and inflammatory diseases. Therefore, acombination consisting of an agonist of GC-C with an inhibitor ofcGMP-PDE might produce synergistic effect on levels of cGMP in thetarget tissues and organs.

Sulindac Sulfone (SS) and Zaprinast (ZAP) are two of the knowninhibitors of cGMP-PDE and have been shown to induce apoptosis in cancercells via a cGMP-dependent mechanism. SS and ZAP in combination withSP-304 or SP-333 were evaluated to see if these PDE inhibitors had anysynergistic effects on intracellular accumulation of cGMP (FIG. 9-12).As the data show, SS at a concentration of 100 μM did not enhanceintracellular accumulation of cGMP. However, the combination of SS withSP-304 stimulated cGMP production several-fold more then stimulation bySP-304 alone. This synergistic effect on cGMP levels was more pronouncedwhen SP-304 were used at a 0.1 μM concentration (FIG. 10). Similarobservations were made when SP-304 or SP-333 were used in combinationwith ZAP (FIG. 10, FIG. 11 and FIG. 12). These results suggest that theintracellular levels of cGMP are stabilized because SS inhibits cGMP-PDEthat might be responsible for depletion of intracellular cGMP. Thus, theapproach to use a combination of GCC agonist with a cGMP-PDE inhibitoris attractive.

For the results shown in FIG. 9, cyclic GMP synthesis in T84 cells wasassessed essentially as described in Shailubhai et al., Cancer Research60, 5151-5157 (2000). Briefly, confluent monolayers of T-84 cells in24-well plates were washed twice with 250 μl of DMEM containing 50 mMHEPES (pH 7.4) and pre-incubated at 37° C. for 10 minutes with 250 μl ofDMEM containing 50 mM HEPES (pH 7.4) and 1 mM isobutyl methylxanthine(IBMX). Monolayers of T84 cells were then incubated with 250 μl of DMEMcontaining 50 mM HEPES (pH 7.4) containing SP-304 or PDE inhibitorseither alone or in combinations, as indicated below in the followingexperimental sets: 1) Control; 2) SP-304 (0.1 μM); 3) Sulindac Sulfone(100 μM); 4) Zaprinast (100 μM); 5) SP-304 (0.1 μM)+Sulindac Sulfone(100 μM); and 6) SP-304 (0.1 μM)+Zaprinast (100 μM). After the 30 minincubation, the medium was aspirated and the reaction was terminated bythe addition of 3% perchloric acid. Following centrifugation and theaddition of NaOH (0.1 N) to neutralize the pH, intracellular cGMP levelswere determined in lysates using a cGMP ELISA kit (Cat. No. 581021;Cayman Chemical, Ann Arbor, Mich.). Incubations were performed induplicate, and each sample was run in duplicate using the ELISA test.

For the results shown in FIG. 10, the method used was same as the oneused for FIG. 9 except that the monolayers of T84 cells were incubatedwith 500 μl of DMEM containing 50 mM HEPES (pH 7.4) containing SP-304(0.1 or 1.0 μM) or increasing concentrations of PDE inhibitors (0 to 750μM) either alone or in combination with SP-304. After the 30 minincubation, the medium was aspirated and the reaction was terminated bythe addition of 3% perchloric acid. Following centrifugation and theaddition of NaOH (0.1 N) to neutralize the pH, intracellular cGMP levelswere determined in lysates using a cGMP ELISA kit (Cat. No. 581021;Cayman Chemical, Ann Arbor, Mich.). Samples were run in triplicate usingthe ELISA test.

For the results shown in FIG. 11, the method used was same as the oneused for FIG. 10 except that the monolayers of T84 cells were incubatedwith 500 μl of DMEM containing 50 mM HEPES (pH 7.4) containing SP-3333(0.1 or 1.0 μM) or increasing concentrations of ZAP (0 to 500 μM) eitheralone or in combination with SP-333. After the 30 min incubation, themedium was aspirated and the reaction was terminated by the addition of3% perchloric acid. Following centrifugation and the addition of NaOH(0.1 N) to neutralize the pH, intracellular cGMP levels were determinedin lysates using a cGMP ELISA kit (Cat. No. 581021; Cayman Chemical, AnnArbor, Mich.). Samples were run in triplicate using the ELISA test.

For the results shown in FIG. 12, the method used was same as the oneused for FIG. 10 except that the monolayers of T84 cells were incubatedwith 500 μl of DMEM containing 50 mM HEPES (pH 7.4) containing SP-333(0.1 μM) or increasing concentrations of Sulindac Sulfone (0 to 500 μM)either alone or in combination with SP-333. After the 30 min incubation,the medium was aspirated and the reaction was terminated by the additionof 3% perchloric acid. Following centrifugation and the addition of NaOH(0.1 N) to neutralize the pH, intracellular cGMP levels were determinedin lysates using a cGMP ELISA kit (Cat. No. 581021; Cayman Chemical, AnnArbor, Mich.). Samples were run in triplicate using the ELISA test.

Example 7: A Repeated Oral Dose Toxicity Study of SP-304 in CynomolgusMonkeys

The primary purpose of this experiment was to evaluate the toxicity andpharmacokinetics of a repeated oral dose of SP-304 in cynomolgusmonkeys. Treatment with a daily dose of 250 mg of SP-304 for 14consecutive days was well tolerated by all of the monkeys, however thetreatment consistently produced liquid feces and watery diarrhea (FIG.14). Monkeys returned to normal stool consistency within 24-48 hoursfollowing the last dose of SP-304.

Example 8: SP-304 Treatment Improves Stool Consistency and ClearsTNBS-Induced Intestinal Blockage in a TNBS-Induced Murine Model ofColitis

SP-304 is a superior analog of uroguanylin and an agonist of GC-C. Theanal administration of trinitrobenzene sulphonic acid (TNBS) istypically used to produce intestinal blockage, resulting in poor stoolconsistency. As shown in FIG. 13, oral administration of SP-304considerably improved stool consistency in mice treated with TNBS.Treatment with SP-304 at a dose between 0.05 to 0.5 mg/kg body weightwas sufficient to completely restore the consistency score to the levelobserved in mice treated with phosphate buffer instead of TNBS (minusTNBS control). Sulfasalazine, a FDA approved drug used as a positivecontrol, also restored normal stool consistency.

Example 9: A Randomized, Double-Blind, Placebo-Controlled, Single-,Ascending-, Oral-Dose Safety, Tolerability, and Pharmacokinetic Study ofSP-304 in Healthy Adult Human Male and Female Volunteers

The objectives of this study were to assess the safety andpharmacokinetics of a single oral dose of SP-304 in healthy subjects.This was a phase 1, single-site, randomized, double-blind,placebo-controlled, single-, ascending-, oral-dose, sequential doseescalation study of SP-304 in fasted, healthy male and female subjects.A total of 9 cohorts utilizing 8 subjects per cohort (6 SP-304; 2placebo) were utilized, totaling 71 volunteers administered drug (onevolunteer dropping out). Each cohort was administered a single, oraldose or matching placebo administered in 10-fold diluted phosphatebuffered saline (PBS) (240 mL). Subjects were only administered one doseof study treatment and could not be enrolled in subsequent cohorts. Thenine cohort doses included 0.1, 0.3, 0.9, 2.7, 5.4, 8.1, 16.2, 24.3 mgand 48.6 mg SP-304.

Doses of SP-304

-   -   0.1 mg (6 active, 2 placebo)    -   0.3 mg (6 active, 2 placebo)    -   0.9 mg (6 active, 2 placebo)    -   2.7 mg (6 active, 2 placebo)    -   5.4 mg (6 active, 2 placebo)    -   8.1 mg (6 active, 2 placebo)    -   16.2 mg (5 active, 2 placebo)    -   24.3 mg (6 active, 2 placebo)    -   48.6 mg (6 active, 2 placebo)

The decision to proceed to the next cohort was made by the study sponsorand principal investigator after reviewing the preliminary blinded,safety information from the cohort. All safety data collected throughthe 48 hours after dosing were reviewed to assess tolerability of thedose level. A minimum of 3 evaluable subjects were required for thedetermination of safety and tolerability at each dose level.

The stopping criteria were: 1) clinically significant adverse events[including clinically significant changes in laboratory orelectrocardiogram (ECG) parameters] in >4 subjects (collectively withina cohort), or 2) 1 drug related, serious adverse event (SAE). No higherdoses were to be administered if one of these criteria was met.Otherwise, the study could proceed to the next higher dose cohort.

Safety was monitored by physical examinations, vital signs, clinicallaboratory tests (hematology, chemistry, urinalysis, fecal occultblood), ECG, and adverse experience assessments). Serial blood sampleswere collected 0, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 16, 24, 36, and 48 hoursafter dosing. Plasma samples were assayed by a validated method forSP-304, and pharmacokinetic parameters calculated. Pharmacodynamicendpoints that were evaluated included time to first stool, stoolfrequency (48-hour period), and stool consistency (48-hour period) usingthe Bristol Stool Form Scale (BSFS).

The phase 1 study (Protocol No. SP-SP304101-08) used an oral solutionprepared by a registered licensed pharmacist at the investigation sitenot more than one hour before administration of dose.

The primary objectives of this clinical evaluation were to determinesafety, toxicity and systemic absorption of a single oral dose ofSP-304. The data indicated that SP-304 was well-tolerated at all dosagelevels and there were no severe adverse events (SAEs). The mostprevalent adverse event (AE) observed during this study was grade Idiarrhea (12.7%), as defined using the Common Terminology Criteria forAdverse Events (CTCAE), which is an increase in the number of bowelmovements from 1 and <4 in a 24-hour period. Notably, SP-304 wasexpected to promote bowel movement, thus the increase in number of bowelmovements was considered to be related to the pharmacodynamic (PD)action of SP-304.

The effect of a single oral dose of SP-304 on stool consistency, asjudged by the Bristol form stool scale (BSFS), was also examined involunteers. The BSFS score for the seven types of stool are:

-   -   Type 1: Separate hard lumps, like nuts (hard to pass)    -   Type 2: Sausage-shaped, but lumpy    -   Type 3: Like a sausage but with cracks on its surface    -   Type 4: Like a sausage or snake, smooth and soft    -   Type 5: Soft blobs with clear cut edges (passed easily)    -   Type 6: Fluffy pieces with ragged edges, a mushy stool    -   Type 7: Entirely liquid

Types 1 and 2 indicate constipation, with 3 and 4 being the “idealstools” especially the latter, as they are the easiest to pass, and 5-7score indicate further tending towards diarrhea or urgency.

FIG. 15A-B shows the effect of a single dose of SP-304 or placebo onBSFS score in volunteers treated with SP-304 ranging from 0.1 mg up to amaximum of 48.6 mg dose. The data indicate that treatment with SP-304produced an increase in BSFS score in volunteers, relative toplacebo-treated volunteers, reflecting a change in stool consistencytowards a looser bowel movement in SP-304 treated volunteers. Theseresults indicate that SP-304 has the potential to normalize bowelmovement and to relieve the discomfort due to chronic constipation.

FIG. 16 shows the effect of a single dose of SP-304 or placebo on thetime to first stool in the 24 hours period following dosing. The dataindicate that SP-304 treatment significantly decreased the time to firstbowel movement from 10.6 hours in volunteers treated with placebo toabout 3 to 6 hours, following SP-304 treatment at doses ranging from 2.7to 48.6 mg.

Example 10: SP-304 Ameliorates Inflammation in DSS-Induced Colitis inBDF-1 Mice

The cGMP pathway mediates the anti-inflammatory effects of cellularmolecules such as nitric oxide and heme oxygenase-1. Therapies thatinduce cGMP (phosphodiesterase-4 inhibitors) have demonstrated efficacyin murine models of IBD. The anti-inflammatory effects of the GCCagonist SP-304 were evalated in a murine model of ulcerative colitis,the DSS-induced colitis model.

Forty eight BDF1 mice were divided into 8 treatment groups (6mice/group). One group was not exposed to DSS (untreated control) andgroups 2-10 were treated with 5% DSS in the drinking water. DSS wasrefreshed daily. All mice were weighed on day −1, and treated with thetest materials beginning on day −1. 4 hrs post dosing on Day 0, DSS wasplaced in the drinking water of groups 2-8 and DSS remained in the wateruntil the end of the study. The test agents were administered at 9 AMdaily until day 7. Animals were treated with a single dose of testagents and the groups were as follows:

-   -   1. No DSS exposure−PBS gavage (No DSS control)    -   2. 5% DSS+PBS (Vehicle control)    -   3. 5% DSS+80 mg/kg Sulfasalazine (positive control)    -   4. 5% DSS+0.005 mg/kg SP-304    -   5. 5% DSS+0.05 mg/kg SP-304    -   6. 5% DSS+0.5 mg/kg SP-304    -   7. 5% DSS+2.5 mg/kg SP-304    -   8. 5% DSS+50 mg/kg SP-304

All doses were administered by oral gavage using a 0.1 ml dose per 10 gbody weight. To avoid cage-to-cage variation, different treatment groupswere housed in the same cage and animals were ear punched foridentification purposes. Mice were sacrificed on day 7, 4-6 hrs postlast dosing. The animals were also subjected to internal examination ofthe major organs for any gross abnormalities. The distal section of thelarge intestine (sufficient for histopathological examination) wasremoved and fixed in Carnoy's solution and embedded in paraffin. Twonon-serial sections per slide were cut and H&E stained for visualseverity score analysis. All slides were scored in a blinded manner.

Histopathology scoring 0 normal 1 all crypts remaining but lookabnormal, all muscle intact 2 less than 90% crypts remaining, all muscleintact 3 less than 75% crypts remaining, majority muscle intact 4 lessthan 10% crypts remaining, most of muscle degraded 5 no crypts left,muscle degraded

Five different sections of the tissue were examined forhistopathological scoring and the scores were averaged for each mouse.The histopathology scores in FIG. 17 are expressed as an average of 6mice. As shown in FIG. 17, the data indicate that treatment of mice withDSS produced mild inflammation in the large intestine. As expected, theseverity of the inflammation was considerably reduced in mice treatedwith sulfasalazine. Similarly, mice treated with SP-304 doses rangingfrom 0.005 to 5 mg/kg/body weight also showed reduced inflammation inthe colon tissue. These results indicate that oral administration withSP-304 ameliorated DSS-induced inflammation in the colon tissue. Thetreatment with SP-304 did not change the colon weight considerably.

Example 11: SP-304 Ameliorates Inflammation in TNBS-Induced Colitis inBDF-1 Mice

Anal administration of TNBS is widely used to induce inflammation in thecolon of mice and rats. The TNBS-induced ulcerative colitis is commonlyused model for experimental colitis in mice for evaluation of drugs tobe used for treatment of IBD in humans. To evaluate theanti-inflammatory effects of the GCC agonist SP-304, ninety BDF-1 micewere randomly divided into 9 groups of 10 each as follows:

-   -   1. No TNBS exposure−PBS gavage (No TNBS control)    -   2. TNBS+PBS (Vehicle control)    -   3. TNBS+80 mg/kg Sulfasalazine (positive control)    -   4. TNBS+0.0005 mg/kg SP-304    -   5. TNBS+0.005 mg/kg SP-304    -   6. TNBS+0.05 mg/kg SP-304    -   7. TNBS+0.5 mg/kg SP-304    -   8. TNBS+2.5 mg/kg SP-304    -   9. TNBS+50 mg/kg SP-304

Groups 2-9 were given 2.5 mg of TNBS in 50% ethanol through anal routeusing a rubber catheter on day 0. Mice were given a single dose ofSP-304 at 9 am everyday for seven days. At the end of the study micewere sacrificed by cervical dislocation. The distal large intestine wasremoved and fixed in Carnoy's fixative. Samples were paraffin embeddedand 2 non-serial sections per sample were cut & mounted on one slidebefore staining with H&E. Slides of intestinal tissues was scored.Blinded histological sections were observed microscopically and assigneda severity score of 0 to 5, as per the scoring system described in FIG.8. For every mouse 5 cross sectional areas of the large intestine wereassessed. Results are expressed as an average. As shown in FIG. 18,treatment with SP-304 at a dose as low as 0.05 mg/kg body weightsignificantly reduced colonic inflammation. Interestingly, the potencyof SP-304 even at concentrations as low as 0.05 mg·kg was comparable tosulfasalazine given at a dose of 80 mg/kg body weight.

Example 12: Repeated Daily Dose of SP-304 Produced Severe Diarrhea inCynomolgus Monkeys

Male (n=4) and female (n=4) monkeys were given a daily dose (1 or 10 or75 mg/kg body weight) of SP-304 repeatedly for 28 days. Effect oftreatment on stool consistency was recorded three times a day. As shownin FIG. 19, oral treatment with SP-304 produced diarrhea/watery stoolsin both sexes. However, female monkeys showed a more pronounced effect.In females, a dose of 10 mg/kg body weight produced severe diarrheaconsistently. Therefore, SP-304 was used at 10 mg/kg body weight in thesubsequent experiments. Similar results were obtained with SP-333.

Example 13: Repeated Dose of SP-304 Produced Severe Bloating in theProximal Intestines in Mice

The objective of this experiment was to determine the primary site ofaction for orally administered SP-304 with respect to its ability tostimulate water secretion in the gastrointestinal tract. Under normalphysiological circumstances, water secretion occurs primarily in theduodenum and the secreted water is then reabsorbed in the ileum. Mice(females, n=6; males, n=6) were given a single dose of SP-304 by oralgavage and sacrificed 30 minutes later. The gastrointestinal tract wasexamined for signs of bloating which indicates excessive secretion ofwater. As shown in Table VIII, SP-304 produced bloting only in thestomach and in the proximal intestine (duodenum and jejunum) but not incecum or distal intestine (ileum and colon). These results demonstratethat orally administered SP-304 caused water secretion in theduodenum/jejunum. Thus, the site of action of SP-304 is primarily in theduodenum and jejunum portions of the gastrointestinal tract.

TABLE VIII SP-304 oral administration produced severe bloating inproximal intestine of mice. Mice (6 males and 6 females) were orallyadministered with SP-304 (1200 mg/kg body weight). After 30 minutes,mice were sacrificed and immediately opened to determine if SP-304administration had caused bloating, due to excessive secretion of fluid,in different segments of the GI tract. Results are expressed as % numberof mice showing bloating in various parts of the GI tract. Male MiceFemale Mice Number of Animals Number of Animals with Bloating withBloating GI Tract Segment (% of total) n = 6 (% of total) n = 6 Stomach3 (50%) 2 (33%) Duodenum 2 (33%) 2 (33%) Jejunum 6 (100%) 6 (100%) Cecum1 (2%) 0 (0%)

Example 14: Formulations of SP-304 for Different GI Diseases

As indicated by the data in Table VIII, orally administered SP-304 actsin the proximal portions of the GI tract (duodenum, jejunum) tostimulate water secretion. Thus, a formulation for delivery of SP-304 tothis region should demonstrate improved efficacy for the treatment ofchronic constipation, IBS-C and other diseases of the proximalintestine. This is because such a formulation would more effectivelystimulate the secretion of water and promote the normalization of bowelmovement in patients suffering from these conditions. In addition,aggregation of SP-304, which occurs beginning at 1.0 mg/ml and ispromoted by acidic conditions, would be minimized in a pH dependentrelease formulation designed to release at higher pH. Thus, a pHdependent release formulation of SP-304 comprising the Eudragit® polymerwas tested for efficacy of release at pH greater than 5.5, which wouldtarget release to the duodenum. As shown in FIG. 20, gelatin capsulescoated with Eudragit® polymer for dissolution at pH greater than 5.5 didnot disintegrate and SP-304 was not released under acidic conditions atpH 1 or 2.5. As expected, incubation of the capsule at pH 5.7 releasedSP-304 within 20 minutes and within 60 minutes most of the peptide wasreleased. The released SP-304 was biologically active as determined inthe T84 cells bioassay (see FIG. 21).

For the treatment of IBD and other diseases or disorders of the distalGI tract, it is advantageous to develop a formulation which targets GCCagonists to the distal GI tract, particularly the terminal ileum. Thisis particularly the case for the treatment of IBD which is oftencomplicated by diarrhea. Thus, oral administration of a GCC agonistwould likely be counterproductive for IBD due to the stimulation ofwater secretion in the duodenum. This problem would be circumvented by aformulation that targeted delivery to the terminal ileum. A pH dependentrelease formulation of SP-304 was therefore tested for efficacy ofrelease at pH greater than 7, which would target release to the terminalileum. As shown in FIGS. 20 and 21, the Eudragit® polymer formulationreleased the SP-304 at pH 7.2 and the released SP-304 was biologicallyactive.

Example 15: SP-304 and SP-333 Formulated in Eudragit® Polymer Coatingfor Delivery at or Above pH 7 Minimized Diarrhea in Cynomolgus Monkeys

As shown in FIG. 22, SP-304 formulated in gelatin capsules coated withEudragit® polymer (for dissolution at pH above 7) produced considerablyless incidences of diarrhea as compared to the uncoated capsulescontaining the same dose of SP-304 (10 mg/kg body weight). These resultsdemonstrate that the delivery of a GCC agonist to the distal intestinereduces the incidence of diarrhea which would otherwise be expected fromoral administration of the agonist. Thus, such a formulation would bepreferred for the treatment of IBD, colon cancer and other diseases ofthe distal intestine.

SP-333 is a GCC agonist which was designed for increased stabilityagainst the proteolysis which would normally occur in the intestinalfluid. Thus, this peptide would also be useful for the treatment of IBD,colon cancer and other diseases of the distal intestine. SP-333 wasformulated in gelatin capsules coated with Eudragit® polymer fordissolution at pH above 7. As shown in FIG. 23, the coated capsulesproduced a considerably lower incidence of diarrhea compared to theuncoated capsules.

1-24. (canceled)
 25. A method for treating constipation in a subject inneed thereof, comprising administering to the subject a GCC agonistformulation comprising (1) a core, which contains at least one GCCagonist peptide, and (2) pH-dependent polymer which degrades in a pHrange of 4.5 to 5.5 or in a pH range of 5.5 to 6.5, wherein theformulation is optimized for delivery of the GCC agonist peptide to theduodenum or jejunum, wherein the GCC agonist peptide is selected fromthe group consisting of SEQ ID NOs: 1-44 and 99-107, and wherein uponadministration to a subject in need thereof, said formulation results infewer unwanted side effects compared to a formulation not optimized fordelivery the duodenum or jejunum. 26-33. (canceled)
 34. The method ofclaim 25, further comprising administering to the subject an effectiveamount of an inhibitor of a cGMP-specific phosphodiesterase. 35.(canceled)
 36. The method of 25, further comprising administering to thesubject an effective amount of at least one laxative.
 37. (canceled) 38.The method of claim 25, further comprising administering to the subjectan effective amount of at least one anti-inflammatory agent.
 39. Themethod of claim 25, wherein the subject is a human.